Dengue virus

I. Organism Information

A. Taxonomy Information
  1. Species:
    1. Dengue virus type 1 :
      1. GenBank Taxonomy No.: 11053
      2. Description: Dengue fever and dengue hemorrhagic fever (DF/DHF) are caused by the dengue viruses, which belong to the genus Flavivirus, family Flaviviridae. There are four antigenically related, but distinct, dengue virus serotypes (DEN-1, DEN-2, DEN-3 and DEN-4), all of which can cause DF/DHF (Gubler, 1997). All analyses undertaken to date show that the four serotypes of dengue virus are phylogenetically distinct, and often to the same degree as different "species" of flaviviruses (Holmes and Twiddy, 2003). The first dengue viruses were isolated from soldiers who became ill in Calcutta, India, New Guinea, and Hawaii. The viruses from India, Hawaii, and one strain from New Guinea were antigenically similar, whereas three other strains from New Guinea appeared to be different. They were called dengue 1 (DEN-1) and dengue 2 (DEN-2) and designated as prototype viruses (DEN-1, Hawaii and DEN-2, New Guinea-C) (Gubler, 1988).
      3. Variant(s):
        • Dengue virus type 1 (strain 924-1) :
          • GenBank Taxonomy No.: 11056
          • Parent: Dengue virus type 1
          • Description: Four DEN-1 virus strains were isolated from humans with classical DF: AHF82-80 (Thailand 1980), 836-1 (Philippines 1984, strain 162, AP2), 924-1 (Mexico 1983, strain 1378) and CV1636/77 (Jamaica 1977) (Chu et al., 1989).
        • Dengue virus type 1 (strain TH-SMAN) :
          • GenBank Taxonomy No.: 31633
          • Parent: Dengue virus type 1
          • Description: TH-36 was isolated in 1958 by Hammon and co-workers from a patient with DHF in Bangkok. TH-Sman was isolated from a similar patient by Dr. Sman Vardhanabhuti (Shiu et al., 1992).
        • Dengue virus type 1 (strain Western Pacific) :
          • GenBank Taxonomy No.: 11059
          • Parent: Dengue virus type 1
          • Description: Western Pacific strain (West Pac) of DEN-1, isolated from a patient with a mild case of DF in 1974 (Puri et al., 1998). The parent DEN-1 strain used in these studies, 45AZ5 PDK-0, was derived from the human isolate, West Pac 74, made from a mild case of dengue fever during an outbreak on Nauru Island in the Western Pacific in 1974 (Puri et al., 1997).
    2. Dengue virus type 2 :
      1. GenBank Taxonomy No.: 11060
      2. Description: The first dengue viruses were isolated from soldiers who became ill in Calcutta, India, New Guinea, and Hawaii. The viruses from India, Hawaii, and one strain from New Guinea were antigenically similar, whereas three other strains from New Guinea appeared to be different. They were called dengue 1 (DEN-1) and dengue 2 (DEN-2) and designated as prototype viruses (DEN-1, Hawaii and DEN-2, New Guinea-C) (Gubler, 1988).
      3. Variant(s):
        • Dengue virus type 2 (isolate Malaysia M1) :
          • GenBank Taxonomy No.: 11061
          • Parent: Dengue virus type 2
          • Description: Dengue-2 (DEN-2) viruses, MI, M2 and M3, isolated in Malaysia from patients with dengue haemorrhagic fever, dengue shock syndrome and dengue fever, respectively (Fong et al., 1990).
        • Dengue virus type 2 (isolate Malaysia M2) :
          • GenBank Taxonomy No.: 11062
          • Parent: Dengue virus type 2
          • Description: Dengue-2 (DEN-2) viruses, MI, M2 and M3, isolated in Malaysia from patients with dengue haemorrhagic fever, dengue shock syndrome and dengue fever, respectively (Fong et al., 1990).
        • Dengue virus type 2 (isolate Malaysia M3) :
          • GenBank Taxonomy No.: 11063
          • Parent: Dengue virus type 2
          • Description: Dengue-2 (DEN-2) viruses, MI, M2 and M3, isolated in Malaysia from patients with dengue haemorrhagic fever, dengue shock syndrome and dengue fever, respectively (Fong et al., 1990).
        • Dengue virus type 2 (strain 16681-PDK53) :
        • Dengue virus type 2 (strain TH-36) :
          • GenBank Taxonomy No.: 31637
          • Parent: Dengue virus type 2
          • Description: TH-36 was isolated in 1958 by Hammon and co-workers from a patient with DHF in Bangkok (Shiu et al., 1992). In 1958 an epidemic of hemorrhagic fever occurred in and near Bangkok, Thailand during the rainy season. The disease there was called Thai hemorrhagic fever. Over 2500 patients were hospitalized with about 10% case fatality rate. During the epidemic, Hammon et al. isolated several viruses both from human sera and from Aedes aegypti. Among these isolates a prototype strain named TH-36 (representing a number of apparently identical isolates) was found to be antigenically closely related to dengue type 2 (Ibrahim et al.,1968).
    3. Dengue virus type 3 :
      1. GenBank Taxonomy No.: 11069
      2. Description: The first dengue viruses were isolated from soldiers who became ill in Calcutta, India, New Guinea, and Hawaii. The viruses from India, Hawaii, and one strain from New Guinea were antigenically similar, whereas three other strains from New Guinea appeared to be different. They were called dengue 1 (DEN-1) and dengue 2 (DEN-2) and designated as prototype viruses (DEN-1, Hawaii and DEN-2, New Guinea-C). Two more serotypes-dengue 3 (DEN-3) and dengue 4 (DEN-4)-were subsequently isolated from patients with a hemorrhagic disease during an epidemic in Manila in 1956 (Gubler, 1988).
    4. Dengue virus type 4 :
      1. GenBank Taxonomy No.: 11070
      2. Description: The first dengue viruses were isolated from soldiers who became ill in Calcutta, India, New Guinea, and Hawaii. The viruses from India, Hawaii, and one strain from New Guinea were antigenically similar, whereas three other strains from New Guinea appeared to be different. They were called dengue 1 (DEN-1) and dengue 2 (DEN-2) and designated as prototype viruses (DEN-1, Hawaii and DEN-2, New Guinea-C). Two more serotypes-dengue 3 (DEN-3) and dengue 4 (DEN-4)-were subsequently isolated from patients with a hemorrhagic disease during an epidemic in Manila in 1956 (Gubler, 1988).
B. Lifecycle Information :
  1. Virion :
    1. Size: 40 to 60 nm in diameter, containing an electron dense core (about 30 nm in diameter) surrounded by a lipid bilayer (Lindenbach and Rice, 2001).
    2. Shape: Dengue viruses are spherical, lipid-enveloped viruses (Guzman and Kouri, 2004).
    3. Picture(s):
      1. Dengue 2 virus particles (Pacific Center for Emerging Infectious Diseases Research):



        Description: Mature particles of Dengue-2 replicating in tissue culture (Pacific Center for Emerging Infectious Diseases Research).
    4. Description: Flavivirus virions consist of a spherical ribonucleoprotein core surrounded by a lipoprotein envelope with small surface projections. The projections seen in electron micrographs are clarified by x-ray crystallography and represent molecules of envelope glycoprotein, which form rodlike structures anchored to the viral membrane at their basal ends. Envelope lipids constitute about 17% of the virion dry weight and are derived from the host cell lipids (Burke and Monath, 2001).

  2. Description: Dengue virus is transmitted in a cycle involving humans and mosquitoes, Aedes aegypti being the most important vector (Burke and Monath, 2001). Two distinct transmission cycles have been described for DENV: Endemic and epidemic cycles that occur in urban/periurban environments and involve human reservoir and amplification hosts. The peridomestic mosquito Ae. aegypti is the principal DENV vector, with Ae. albopictus and other anthropophilic Aedes mosquitoes serving as secondary vectors. Ecologically distinct, sylvatic, enzootic cycles of DENV occur in west Africa and Malaysia, probably involving non-human primate reservoir hosts and sylvatic Aedes spp. mosquito vectors. The two kinds of DENV cycles are also evolutionarily distinct, and all four serotypes of endemic/epidemic DENV are believed to have evolved independently from sylvatic progenitors during the past few 1,000 years (Diallo et al., 2005).
  3. Transmission of Dengue Virus by Aedes aegypti (CDC Dengue Slideset):



    Description: The transmission cycle of dengue virus by the mosquito Aedes aegypti begins with a dengue-infected person. This person will have virus circulating in the blood-a viremia that lasts for about five days. During the viremic period, an uninfected female Aedes aegypti mosquito bites the person and ingests blood that contains dengue virus. Although there is some evidence of transovarial transmission of dengue virus in Aedes aegypti, usually mosquitoes are only infected by biting a viremic person. Then, within the mosquito, the virus replicates during an extrinsic incubation period of eight to twelve days. The mosquito then bites a susceptible person and transmits the virus to him or her, as well as to every other susceptible person the mosquito bites for the rest of its lifetime. The virus then replicates in the second person and produces symptoms. The symptoms begin to appear an average of four to seven days after the mosquito bite-this is the intrinsic incubation period, within humans. While the intrinsic incubation period averages from four to seven days, it can range from three to 14 days. The viremia begins slightly before the onset of symptoms. Symptoms caused by dengue infection may last three to 10 days, with an average of five days, after the onset of symptoms-so the illness persists several days after the viremia has ended (Source: CDC) (CDC Dengue Slideset).
C. Genome Summary:
  1. Genome of Dengue virus type 1 (strain Western Pacific)
    1. Description: The four serotypes of dengue (DEN) virus belong to the genus Flavivirus in the family Flaviviridae. These are single-stranded positive-sense RNA viruses with a genome of about 11000 bases that codes for three structural proteins, C-prM-E; seven nonstructural proteins, NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5; and short non-coding regions on both the 5' and 3' ends (Puri et al., 1997).
    2. Chromosome:
      1. GenBank Accession Number: NC_001477
      2. Size: 10735 bp ss-RNA (NCBI Entrez Genome).
      3. Gene Count: The genome of flaviviurses consists of a single-stranded RNA about 11 kilobases (kb) in length. This RNA contains a 5' cap [m(7)G51ppp5'A) at the 51 end and lacks a polyadenylate tail. Genomic RNA is the messenger RNA for translation of a single long open reading frame (ORF) as a large polyprotein (Lindenbach and Rice, 2001).
      4. Description: The complete nucleotide sequences of the genomes of dengue-1 virus virulent 45AZ5 PDK-O and attenuated vaccine candidate strain 45AZ5 PDK-27 have been determined and compared with the dengue-1 virus Western Pacific (West Pac) 74 parent strain from which 45AZ5 PDK-O was derived. Twenty-five (0.23%) nucleotide and 10 (0.29%) amino acid substitutions occurred between parent strain dengue-1 virus West Pac 74 and virulent strain 45AZ5 PDK-O, which was derived from the parent by serial passage in diploid foetal rhesus lung (FRhL-2) and mutagenized with 5-azacytidine. These substitutions were preserved in the 45AZ5 PDK-27 vaccine. 45AZ5 PDK-O and PDK-27 strains, which differ by 27 passages in primary dog kidney (PDK) cells, show 25 (0.23%) nucleotide and 11 (0.32%) amino acid divergences. These comparative studies suggest that the changes which occurred between the West Pac 74 and 45AZ5 PDK-O strains may alter the biological properties of the virus but may not be important for attenuation. Important nucleotide base changes responsible for attenuation accumulated between 45AZ5 PDK-O and 27 (Puri et al., 1997).

  2. Genome of Dengue virus type 2 (strain PR159/S1)
    1. Chromosome:
      1. GenBank Accession Number: NC_001474
      2. Size: 10703 bp ss-RNA (NCBI Entrez Genome)
      3. Description: We have determined the complete sequence of the RNA of dengue 2 virus (S1 candidate vaccine strain derived from the PR-159 isolate) with the exception of about 15 nucleotides at the 5' end. The genome organization is the same as that deduced earlier for other flaviviruses and the amino acid sequences of the encoded dengue 2 proteins show striking homology to those of other flaviviruses. The overall amino acid sequence similarity between dengue 2 and yellow fever virus is 44.7%, whereas that between dengue 2 and West Nile virus is 50.7%. These viruses represent three different serological subgroups of mosquito-borne flaviviruses. Comparison of the amino acid sequences shows that amino acid sequence homology is not uniformly distributed among the proteins; highest homology is found in some domains of nonstructural protein NS5 and lowest homology in the hydrophobic polypeptides ns2a and 2b. In general the structural proteins are less well conserved than the nonstructural proteins. Hydrophobicity profiles, however, are remarkably similar throughout the translated region. Comparison of the dengue 2 PR-159 sequence to partial sequence data from dengue 4 and another strain of dengue 2 virus reveals amino acid sequence homologies of about 64 and 96%, respectively, in the structural protein region. Thus as a general rule for flaviviruses examined to date, members of different serological subgroups demonstrate 50% or less amino acid sequence homology, members of the same subgroup average 65-75% homology, and strains of the same virus demonstrate greater than 95% amino acid sequence similarity (Hahn et al., 1988).

  3. Genome of Dengue virus type 3
    1. Chromosome:
      1. GenBank Accession Number: NC_001475
      2. Size: 10,696 bp ss-RNA (NCBI Entrez Genome).
      3. Description: The complete nucleotide sequence of the genome of the dengue virus type 3 was determined. Sequence analyses of the genomic RNA and cloned cDNA revealed that the genomic RNA contains 10,696 nucleotides and encodes a single open reading frame of 10,170 nucleotides corresponding to 3390 amino acid residues. The N-terminal amino acid sequences of three structural proteins (C, M, and E proteins) and the preM protein were also determined from the purified virion. When the deduced amino acid sequence and N-terminal amino acid sequence determined from purified proteins were compared with those of other flaviviruses, the genome organization was found to be the same as that of other flaviviruses (Osatomi and Sumiyoshi, 1990).

  4. Genome of Dengue virus type 4
    1. Chromosome:
      1. GenBank Accession Number: NC_002640
      2. Size: 10,649 bp ss-RNA (NCBI Entrez Genome).
      3. Description: Unpublished sequence (NCBI Entrez Genome).

II. Epidemiology Information

The first reported epidemics of dengue-like disease occurred on three separate continents almost simultaneously in 1779 and 1780. Although there is some disagreement as to whether all of these epidemics were caused by dengue viruses, it is clear that dengue and other arboviruses with similar ecology had widespread distribution in the tropics as long as 200 years ago. For the next 175 years major pandemics of dengue-like illness, occurred in Asia and the Americas at variable intervals ranging from 10 to 30 years. With the advent of modern diagnostic virology, and the isolation and identification of the four dengue virus serotypes, their distribution became better known. Asia historically has been the area of highest endemicity, with all four dengue serotypes circulation in the large urban centers of most countries. During and shortly after World War II, Ae. aegypti became more widespread in Asia, and with the subsequent urbanization that occurred in most countries, the incidence of dengue infection increased dramatically. This increase coincided with the emergence of epidemic DHF in the 1950s (Gubler, 1988). The factors responsible for this global resurgence of DF and the emergence of DHF include unprecedented population growth, unplanned and uncontrolled urbanisation, increased air travel, the lack of effective mosquito control, and the deterioration, during the past 30 years, of public health infrastructure (Rigau-Perez et al., 1998). The average number of DF/DHF cases reported to WHO per year has risen from 908 between 1950 and 1959 to 514,139 between 1990 and 1999. The real figure is estimated to be closer to 50 million cases a year causing 24,000 deaths. Of an estimated 500,000 cases of DHF/DSS requiring hospitalisation each year, roughly 5% die according to WHO statistics (Guha-Sapir and Schimmer, 2005).

A. Outbreak Locations:
  1. Asia. Epidemic DF was a common occurrence in Asia in the first 50 years of the 20th century. Epidemic waves would move through the region every 10 to 40 years, depending on when a new virus was introduced. DEN viruses were endemic in many cities of Asia during this time as documented by the numerous accounts of expatriates arriving in a tropical Asian city only to become ill with a severe dengue-like illness within weeks to months of arrival (Gubler, 2004).
  2. Maldives. Maldives has experienced an outbreak of dengue since January 2006, with 602 suspected cases until 5 March 2006 (including 64 cases of dengue haemorrhagic fever and 9 cases of dengue shock syndrome) (Weekly Epidemiological Record, 2006).
  3. India. The new dengue paradigm viz, the burst of sudden disease activity, persistence and diffusion of the disease in different areas has secured a foot-hold in Southern India and has emerged as a serious public health problem. In Tamil Nadu, annual reports of dengue cases and deaths due to dengue were ranging from 128 to 264 and 2 to 21 respectively up to the year 2000. Recently, between October 2001 and January 2002, an epidemic of dengue emerged in Chennai, Tamil Nadu, affecting adults and children; majority of the victims were children less than 15 yrs of age (Kabilan et al., 2005). Between October 2001 and January 2002, there was an epidemic of dengue in Chennai, with a peak in October. The case occurrence was reported to be high among pediatric group. The number of cases confirmed during the study period was considered as the representative of the cases reported (about 700 cases) to the health surveillance system in Chennai (Kabilan et al., 2005). In India, dengue virus activity has been reported in many parts of the country with sudden epidemics over the last few years. Seasonal and cyclic epidemic pattern of dengue is a recent phenomenon in Northern India. The DF, DHF and DSS have spread dramatically in many parts of the country. Though all age groups were affected in these epidemics, the occurrence was high among children more than 6 yr; and few infants also presented symptoms of DHF (Kabilan et al., 2005).
  4. Timor-Leste. As of 28 February 2005, WHO has received reports of 336 hospitalized cases of dengue infection and 22 deaths.Of the 336 cases, 263 had clinical features compatible with dengue haemorrhagic fever (DHF) and the remaining 73 cases were diagnosed as having suspected dengue fever (DF)using WHO standard case definitions. Districts reporting DF/DHF cases are Baucau, Dili, Ermera, Liquica, Maliana, Manatuto and Viqueque, with 76% of the cases reported from Dili. Preliminary laboratory results have identified Dengue 3 as the main circulating strain in this outbreak (Weekly Epidemiological Record, 2005).
  5. Cuba. During the past three decades there have been four major dengue epidemics in Cuba. The first which was widespread throughout Cuba occurred in 1977 and only dengue fever was observed. This epidemic was caused by an American genotype DENV-1 virus. Subsequently, two independent DHF epidemics caused by DENV-2 of Asiatic origin occurred in 1981 throughout Cuba and 1997 in Santiago de Cuba, four and twenty years respectively, after the epidemic caused by DENV-1 (Rodriguez-Roche et al., 2005)
  6. Brazil. The following study was intended to evaluate the occurrence of typical signs and symptoms in the cases of classic dengue and hemorrhagic dengue fever, during the 2001-2002 epidemic in the city of Rio de Janeiro. The authors reviewed 155,242 cases notified to the Information System of Notification Diseases, from January/2001 to June/2002: 81,327 cases were classified as classic dengue and 958 as hemorrhagic dengue fever, with a total of 60 deaths (Casali et al., 2004).
  7. In early 2004, an outbreak of dengue began to spread throughout Indonesia. On 16 February 2004, the Indonesian Ministry of Health declared a national DF/DHF epidemic. Jakarta, the capital city with approximately 16 million inhabitants, was the most affected area (Suwandono et al., 2006) The Indonesian Ministry of Health reported cases of DF in 30 of 32 providences within the archipelago. In the capital city of Jakarta, a total of 20 503 cases were recorded, with an epidemic peak between March and April (Suwandono et al., 2006).
  8. Palau. An epidemic of dengue 4 virus occurred in the island nation of Palau between January and July 1995. The last known epidemic of dengue in the Palau Islands occurred in 1988 when dengue type 2 was introduced. Before that, dengue transmission had not been reported since 1944. In 1995, higher than expected rainfall for January and February may have contributed to a large mosquito population and increased transmission of the virus. Increased rainfall has been reported previously to be associated with epidemic dengue fever (Ashford et al., 2003). During January and February 1995, 145 patients (an unusually high number) with viral syndrome were reported to the Palau Ministry of Health. On April 3, 1995, a 38-year-old man died at the Palau National Hospital soon after presentation with viral syndrome. He was noted to have had neutropenia and thrombocytopenia. Initially, an outbreak of leptospirosis was suspected (and later this patient was confirmed to have leptospirosis by immunohistochemical analysis). However, the majority of the initial serum samples from patients with febrile illness tested at the Centers for Disease Control and Prevention (CDC) Dengue Laboratory in San Juan, Puerto Rico were consistent with dengue virus infection, suggesting an outbreak of dengue fever (Ashford et al., 2003).
B. Transmission Information:
  1. From: Mosquito To: Human
    Mechanism: After the mosquito becomes infective, it may transmit dengue by taking a blood meal, or by simply probing the skin of a susceptible person (Rigau-Perez et al., 1998)

  2. From: Human To: Mosquito
    Mechanism: The mosquito becomes infected by a blood meal from a viraemic person and becomes infective after an obligatory extrinsic incubation period of 10-12 days (Rigau-Perez et al., 1998).

  3. From: Human To: Human
    Mechanism: Vertical transmission of dengue virus has been recorded in a small number of cases, leading to neonatal DF or even DSS. One case of nosocomial transmission from a needlestick injury has been reported (Rigau-Perez et al., 1998). The vertical transmission of dengue has been infrequently described world-wide, although there are reports from Cuba, Brazil, Malaysia, and Thailand which have occurred during outbreaks. These report variable neonatal outcomes, from asymptomatic infection to death (Perret et al., 2005).

  4. From: Mosquito To: Mosquito
    Mechanism: It was originally thought that all vertical transmission of arboviruses in mosquitoes was transovarial in nature because viral antigen has been demonstrated in developing eggs for bunyaviruses, and both bunyaviruses and flaviviruses have been recovered from progeny reared from surface-sterilized eggs. It was eventually discovered, however, that vertical transmission of dengue viruses, and at least certain other flaviviruses, takes place in the genital chamber of the female as mature eggs are fertilized during oviposition. This explains how vertical transmission of dengue viruses can occur without virus in developing eggs (Rodhain and Rosen, 1997). Progeny of Aedes aegypti mosquitoes infected intrathoracically with dengue-3 virus was reared to subsequent generations. In each generation, blood-fed females were confined individually and the eggs obtained from the transovarially infected females were pooled. The seventh generation obtained from the infected parental mosquitoes showed that virus could persist in mosquitoes in successive generations through transovarial passage. The rate of vertical transmission initially increased in the few generations (F1-F2), but in subsequent generations it was found to be steady (Joshi et al., 2002). These observations, which have great epidemiologic importance, suggest that vector mosquitoes may play an important role in the maintenance of virus in nature, and that mosquitoes may act as reservoirs of these viruses (Joshi et al., 2002). Male Ae. albopictus can transmit dengue virus sexually in the course of mating, and females can transmit it vertically more efficiently than can Ae. aegypti females. These two mechanisms could explain the maintenance of the virus in nature between epidemics in non-endemic areas where susceptible human or primate populations are not always present (Gratz, 2004).

C. Environmental Reservoir:
  1. Humans :
    1. Description: Human dengue viruses are mostly active in urban areas where the virus is maintained through a cycle in which humans are the principal reservoir host and Aedes aegypti is the principal mosquito vector (de Silva et al., 1999).
    2. Survival Information: Dengue infection can cause a spectrum of illness ranging from mild, undifferentiated fever to illness up to 7 days' duration with high fever, severe headache, retro-orbital pain, arthralgia and rash, but rarely causing death (Guha-Sapir and Schimmer, 2005).
  2. Aedes mosquitoes :
    1. Description: The evidence that some Aedes (Stegomyia) species transmit dengue virus vertically suggests that they also could serve as reservoirs of the virus during dry season. their role in the epidemiology of dengue is not known in detail (Rodhain and Rosen, 1997).
    2. Survival Information: Dengue viruses multiply in the midgut epithelium, brain, fat body, and salivary glands of mosquitoes. No detectable pathologic changes result from infection, and mosquitoes remain infectious for life (Burke and Monath, 2001).
  3. Nonhuman Primates :
    1. Description: One or more dengue serotypes, transmitted by Aedes of the niveus group circulate in the forest canopy in primeval cycle among certain species of monkeys (Macaca sp. and Presbytis sp.-which have asymptomatic infections) in a silent cycle. Man is only occasionally involved in this cycle. Such a zoonotic reservoir of infection could exist in all the primary forests of tropical Asia: in Malaysia, in thailand, in Vietnam, in Cambodia, in Indonesia etc (Rodhain, 1991).
    2. Survival Information: There is considerable field evidence from both Malaysia and Africa that lower primates are involved in forest maintenance cycles of dengue viruses. Moreover, experimental laboratory data show that chimpanzees, gibbons, and macaque are susceptible to infection with dengue viruses. All species develop detectable viremia in the absence of clinical illness. The experimental infection data suggest that dengue viruses have become well adapted to lower primates which, therefore, are not useful as laboratory animal models for the study of human disease (Gubler, 1988).
D. Intentional Release:

No release information is currently available here.


III. Infected Hosts

  1. Human:
    1. Taxonomy Information:
      1. Species:
        1. Human :
          • GenBank Taxonomy No.: 9606
          • Scientific Name: Homo sapiens (NCBI Taxonomy)
          • Description: Humans are the major host of dengue virus (Holmes and Twiddy, 2003). There is no consensus on when dengue first appeared in human populations, largely because its symptoms are often not diagnostic. The earliest record suggested is from a Chinese medical encyclopaedia dating to 992 A.D.. However, it is generally agreed that by the late 18th century a disease bearing a strong resemblance to dengue was causing intermittent epidemics in Asia and the Americas, and that by the late 19th and early 20th centuries the virus was probably widespread in the tropics and subtropics. Shortly after World War II, a new dengue-associated disease was reported in endemically infected areas of Southeast Asia. This had a far more pronounced impact than DF, since the primary targets were children. The first well documented outbreak of what came to be known as dengue haemorrhagic fever took place in Manila in 1953/54, and was followed by a larger outbreak in Bangkok in 1958. Since this time DHF/DSS have become endemic in all countries in Southeast Asia, with dramatic increases in case numbers, so much so that dengue is considered an archetypal "emerging" disease (Holmes and Twiddy, 2003).

    2. Infection Process:
      1. Description: Dengue viruses are efficiently transmitted in an urban cycle which involves man and Aedes aegypti, a day-biting species which often breeds in the clean water stored in houses. Infection with one type results in life-long immunity to that type but, after a short period of cross protection, individuals may become clinically ill during and infection with a second type (Halstead, 1988).
        • Aedes aegypti Mosquito (CDC Dengue Slideset):



          Description: The most common epidemic vector of dengue in the world is the Aedes aegypti mosquito. It can be identified by the white bands or scale patterns on its legs and thorax (CDC Dengue Slideset).

    3. Disease Information:
      1. Breakbone Fever (i.e., Dengue Fever (DF), Dengue Hemorrhagic Fever (DHF), Dengue Shock Syndrome (DSS)) :
        1. Pathogenesis Mechanism: Both syndromes, DF and DHF/DSS, are caused by any of the four dengue serotypes that belong to the family Flaviviridae (Guzman and Kouri, 2004). The pathogenesis of DHF/DSS is not very well understood nor are the host conditions that favor the severe disease; however, children, females, individuals with chronic diseases such as asthma and diabetes, and whites appear to be at greater risk. Finally, recent reports argue the risk of DHF/DSS is higher if the interval is longer between primary and secondary dengue infection (Guzman and Kouri, 2004). Although most dengue infections cause only mild clinical disease, understanding the basis of severe dengue disease is an important clinical and scientific goal. Dengue viruses are capable of replicating in many cell types and can be detrimental to cell function. However, the major target for dengue virus infection in vivo appears to be cells of the monocyte/macrophage lineage, in which dengue virus causes little cytopathic effect. It is thought that capillary leakage in DHF results from the release of circulating factors by dengue virus-infected monocytes, activated T cells and other cells. Hemorrhagic manifestations, on the other hand, may be multifactorial due to the direct and indirect effects of dengue virus infection on platelets and the coagulation system. Explanations for the occurrence of severe dengue disease have focused on possible viral and host factors. The available evidence supports the suggestion that severe dengue disease can be more frequently observed with some viral strains than others and that it can occur in the absence of 'enabling' host factors. However, the molecular basis for such an association, if any exists, remains unknown. There is also substantial evidence that the risk of DHF is increased during secondary dengue infections and this immunologic mechanism may predominate in the pathogenesis of capillary leakage. In vitro studies have identified both antibody and T-cell-dependent mechanisms that could exacerbate disease, and clinical studies have correlated the presence of enhancing antibodies and higher levels of T-cell activation with DHF. Thus, both viral and host factors are probably relevant to determining the risk of severe dengue disease, but the interactions and relative importance of all these factors in influencing the expression of clinical disease have not been established (Rothman, 1997).


        2. Incubation Period: The incubation period for dengue is four to six days (Guzman and Kouri, 2004).


        3. Prognosis: The prognosis in DHF/DSS depends on prevention or early recognition and treatment of shock. In hospitals with long experience of DSS the case fatality rate in DHF can be as low as 0.2%. Once shock has set in the fatality rate may be high (12% to 44%) (Rigau-Perez et al., 1998). Dengue infection can cause a spectrum of illness ranging from mild, undifferentiated fever to illness up to 7 days' duration with high fever, severe headache, retro-orbital pain, arthralgia and rash, but rarely causing death. Dengue Haemorrhagic Fever (DHF), a deadly complication, includes haemorrhagic tendencies, thrombocytopenia and plasma leakage. Dengue Shock Syndrome (DSS) includes all the above criteria plus circulatory failure, hypotension for age and low pulse pressure. DHF and DSS are potentially deadly but patients with early diagnosis and appropriate therapy can recover with no sequelae (Guha-Sapir and Schimmer, 2005). The vast majority of infections, especially in children under age 15 years, are asymptomatic or minimally symptomatic. Population-based studies have shown increasing severity in the clinical features of DF with increasing age of the patient and with repeated infections. Infants and young children may have an undifferentiated febrile disease with a maculopapular rash. Older children and adults may have either a mild febrile syndrome or the classical and even incapacitating disease. Skin eruptions are reported in over 50% of laboratory-confirmed dengue cases in Puerto Rico, more commonly in children and adults with primary infections. There may be a flushing of the face, neck, and chest initially in the febrile period; or a centrifugal maculopapular rash arising on the third or fourth day; or a later confluent petechial rash with round pale areas of normal skin; or a combination of these manifestations (Rigau-Perez et al., 1998).


        4. Diagnosis Overview: Dengue diagnosis can be performed through virus isolation, genome and antigen detection and serological studies. Serology is currently the most widely applied in routine diagnosis. Of course, clinical, geographical, and epidemiological data associated with the patient remain critical considerations when evaluating a laboratory result (Guzman and Kouri, 2004). The definitive diagnosis of dengue virus infection can only be made in the laboratory, and it depends on the isolation of these viruses, the detection of viral antigens or RNA in serum or tissues, or the detection of specific antibodies in the patients' serum (De Paula and Fonseca, 2004). Five serological tests have been used for the diagnosis of dengue infection: hemagglutination-inhibition (HI), complement fixation (CF), neutralization test (NT), immunoglobulin M (IgM) capture enzyme linked immunosorbent assay (MAC-ELISA) and indirect immunoglobulin G ELISA. The limitations of these techniques are the high cross-reactivity observed with these tests, requiring a comprehensive pool of antigens, including all four serotypes, another flavivirus (yellow fever virus, Japanese encephalitis virus, or St. Louis encephalitis virus), and in some areas, another virus that causes similar clinical manifestations but that is not flavivirus, such as Oropouche, Mayaro or Chikungunya viruses. Furthermore, the dengue antibodies are better detected around the fifth day of disease onset, making this technique unfeasible for rapid diagnosis (De Paula and Fonseca, 2004)


        5. Symptom Information :
          • Syndrome -- Undifferentiated Fever:
            • Description: Infants and young children usually develop an undifferentiated febrile disease that can be accompanied by a maculopapular rash (Guzman and Kouri, 2004).
          • Syndrome -- Dengue Fever:
            • Description: The clinical features of dengue vary frequently, according to the age of the patient. Infants and young children may have an undifferentiated febrile disease with a maculopapular rash. Older children and adults may have either a mild febrile syndrome or the classical incapacitating disease with abrupt onset and high fever, severe headache, pain behind the eyes, muscle and joint-pains, and rash. Skin hemorrhages (with positive tourniquet test and or/petechiae) may be present. Leukopenia is usually found and thrombocytopenia may be observed. The case fatality rate is exceedingly low (Pan American Health Organization, 1994). Clinical description: An acute febrile illness characterized by frontal headache, retro-ocular pain, muscle and joint pain, and rash (Pan American Health Organization, 1994). Dengue virus infections may be asymptomatic or lead to a range of clinical presentations, even death. The incubation period is 4-7 days (range 3-14). Typically, DF is an acute febrile illness characterised by frontal headache, retroocular pain, muscle and joint pain, nausea, vomiting, and rash. The febrile, painful period of DF lasts 5-7 days, and may leave the patient feeling tired for several more days. A biphasic or "saddleback" fever curve is not the norm. Dengue virus disappears from the blood after an average of 5 days, closely correlated with the disappearance of fever, and no carrier state ensues (Rigau-Perez et al., 1998).
            • Observed: Dengue is the most prevalent mosquito-borne viral infection worldwide, with 100 million cases of dengue fever (DF) and half a million cases of dengue haemorrhagic fever (DHF) annually (Malavige et al., 2006). Today, DF and DHF/DSS are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. More than 2.5 billion people are at risk of infection and more than 100 countries have endemic dengue transmission. DHF has been reported in 60 of them. The burden of DF and DHF disease is not very well documented; however in 1998 alone, more than 1.2 million cases were reported to the World Health Organization, with south-east Asia, the western Pacific and more recently the Americas being the most affected regions (Guzman and Kouri, 2004).


            • Symptoms Shown in the Syndrome:

            • Abdominal pain:
            • Bleeding manifestations:
            • Diarrhoea:
            • Fever:
              • Description: The most severe cases of dengue fever are usually seen in older children and are characterised by a rapidly rising temperature (greater than or equal to 39 C) that lasts 5-6 days (Mairuhu et al., 2004).
              • Observed: 76-100% in Thai adults with classical dengue fever (Pan American Health Organization, 1994).
            • Flushed appearance:
            • Gum bleeding:
              • Description: Minor haemorrhagic manifestations like petechiae, epistaxis, and gingival bleeding do occur (Mairuhu et al., 2004).
              • Observed: 3% in patients with dengue fever had gum bleeding (Malavige et al., 2006).
            • Headache:
              • Description: The febrile period is accompanied by severe headache, reto-orbital pain, myalgia, arthralgia, nausea, and vomiting (Mairuhu et al., 2004).
              • Observed: 78.8% in patients with dengue fever had headaches (Malavige et al., 2006).
            • Hepatomegaly:
            • Leukopenia:
            • Lymphadenopathy:
            • Maculopapular rash:
              • Description: Over half of infected people report a rash during the febrile period that is initially macular or maculopapular and becomes diffusely erythematous, sparing small areas of normal skin ("islands of white in a sea of red") (Mairuhu et al., 2004).
              • Observed: 26-50% in Thai adults with classical dengue fever (Pan American Health Organization, 1994).
            • Myalgia/Arthralgia:
            • Petechiae or ecchymosis:
            • Positive Tourniquet Test:
              • Description: A positive tourniquet test (more than 20 petechiae in a square patch of skin 2.5 x 2.5 cm [greater than 20/in(2)]) may be found in over one-third of patients with DF (Rigau-Perez et al., 1998). The tourniquet test is another stumbling block, because there is confusion in the definition of a positive result (either ten or 20 petechiae per square inch [6.45 cm2]), it is a long test (5 min) for a doctor's visit, and it feels even longer for the patients, since it is very uncomfortable.The omission of the use of the tourniquet test has considerable impact on the detection of dengue haemorrhagic fever. Grade I dengue haemorrhagic fever, which might represent 15-20% of all dengue haemorrhagic fever cases, depends on tourniquet test positivity (Rigau-Perez, 2006).


                • Positive Tourniquet Test (CDC Dengue Slideset):



                  Description: This slide demonstrates what a typical positive result from a tourniquet test may look like. This patient has more than 20 petechiae per square inch. Source: CDC (CDC Dengue Slideset)
              • Observed: 26-50% in Thai adults with classical dengue fever (Pan American Health Organization, 1994).
            • Thrombocytopenia:
            • Vomiting:
              • Description: The febrile period is accompanied by severe headache, reto-orbital pain, myalgia, arthralgia, nausea, and vomiting (Mairuhu et al., 2004).
              • Observed: 54.5% in patients with dengue fever had vomiting (Malavige et al., 2006).
          • Syndrome -- Dengue Hemorrhagic Fever:
            • Description: Today, secondary infection by a different dengue serotype is considered the most significant individual risk factor for DHF/DSS. The presence of circulating non-neutralizing, cross-reactive antibodies in a previously immune individual allows for enhancement of infection, favoring the increased entrance of the virus into the target cell through the cell Fc receptor (Guzman and Kouri, 2004). Clinical Case Definition for Dengue Hemorrhagic Fever. The following must all be present: 1. Fever or recent history of acute fever. 2. Hemorrhagic tendencies, as evidenced by at least one of the following: positive tourniquet test, petechiae, ecchymoses, or purpura; and bleeding from mucosa, gastrointestinal tract, injection sites, or others. 3. Thrombocytopenis [100,000 mm(3) or less]. 4. Plasma leakage due to increased capillary permeability as mainifested by at least one of the following: hematocrit on presentation that is greater than or equal to 20% above average for that age, sex, and population; greater than or equal to 20% drop in hematocrit following treatment; or commonly associated signs of plasma leakage-pleural effusion, ascites, and hypoproteinemia (Pan American Health Organization, 1994). DHF commonly begins with a sudden rise in temperature and other symptoms resembling DF. The temperature is typically high (38-40 C) and continues for 2-7 days. DHF and dengue shock usually develop around the third to seventh day of illness. The most common haemorrhagic feature is a positive tourniquet test (over 50% of patients). Petechiae, easily bruised skin, and subcutaneous bleeding at venepuncture sites are present in most cases. Transudate due to excessive capillary permeability collects at the pleural and abdominal cavities (Rigau-Perez et al., 1998).
            • Observed: Dengue is the most prevalent mosquito-borne viral infection worldwide, with 100 million cases of dengue fever (DF) and half a million cases of dengue haemorrhagic fever (DHF) annually (Malavige et al., 2006). Today an estimated 50-100 million cases of dengue fever and 500,000 cases of DHF, resulting in around 24,000 deaths, occur annually, depending on the epidemic activity (Mairuhu et al., 2004).


            • Symptoms Shown in the Syndrome:

            • Abdominal pain:
              • Description: Several symptoms and signs occur before defervescence and may serve as warning signs that DHF and DSS are impending: generalised abdominal pain, persistent vomiting, change in the level of consciousness, a sudden drop in the platelet count, and a rapid rise in the hematocrit (Mairuhu et al., 2004).
              • Observed: 14% in patients with dengue hemorrhagic fever had abdominal pain (Malavige et al., 2006).
            • Bleeding manifestations:
              • Description: Haemorrhagic manifestation usually appear after 3-4 days and may vary from a positive tourniquet test and petechiae to haemorrhage from the gastrointestinal tract, nose, and gums (Mairuhu et al., 2004).
              • Observed: 49.3% in patients with dengue hemorrhagic fever had bleeding manifestations (Malavige et al., 2006).
            • Confluent petechial rash:
              • Description: Haemorrhagic manifestation usually appear after 3-4 days and may vary from a positive tourniquet test and petechiae to haemorrhage from the gastrointestinal tract, nose, and gums (Mairuhu et al., 2004).
              • Observed: 1-25% in Thai children with classical dengue hemorrhagic fever (Pan American Health Organization, 1994).
            • Diarrhoea:
            • Fever:
            • Flushed appearance:
              • Observed: 44% in patients with dengue hemorrhagic fever had a flushed appearance (Malavige et al., 2006).
            • Gastrointestinal bleeding:
              • Description: Haemorrhagic manifestation usually appear after 3-4 days and may vary from a positive tourniquet test and petechiae to haemorrhage from the gastrointestinal tract, nose, and gums (Mairuhu et al., 2004).
              • Observed: 1-25% in Thai children with classical dengue hemorrhagic fever (Pan American Health Organization, 1994).
            • Gum bleeding:
              • Description: Haemorrhagic manifestation usually appear after 3-4 days and may vary from a positive tourniquet test and petechiae to haemorrhage from the gastrointestinal tract, nose, and gums (Mairuhu et al., 2004).
              • Observed: 6% in patients with dengue hemorrhagic fever had gum bleeding (Malavige et al., 2006).
            • Headache:
              • Description: The febrile period is accompanied by severe headache, reto-orbital pain, myalgia, arthralgia, nausea, and vomiting (Mairuhu et al., 2004).
              • Observed: 60% in patients with dengue hemorrhagic fever had headaches (Malavige et al., 2006).
            • Haematemesis:
              • Observed: 6% in patients with dengue hemorrhagic fever had haematemesis (Malavige et al., 2006). 38% of Puerto Ricans cases confirmed with with dengue hemorrhagic fever in the laboratory (Malavige et al., 2006).
            • Hepatomegaly:
            • Hypotension:
            • Leukopenia:
            • Lymphadenopathy:
            • Melena:
              • Observed: 10.3% in patients with dengue hemorrhagic fever had melena (Malavige et al., 2006). 17% of Puerto Ricans cases confirmed with with dengue hemorrhagic fever in the laboratory (Malavige et al., 2006).
            • Myalgia/Arthralgia:
            • Petechiae or ecchymosis:
            • Pleural effusions or ascites:
              • Description: A prospective study recorded pleural effusions in 84% (22/26) of DHF cases and the mean pleural effusion index (the proportion of the width of the right hemithorax occupied by a pleural effusion in the right lateral decubitus chest radiograph) was 14.1% (Rigau-Perez et al., 1998).


                • Pleural Effusion Index (CDC Dengue Slideset):



                  Description: Here we see a right lateral decubitus X-ray showing a large pleural effusion, typical of DHF the day after defervescence. When the chest X-ray is taken in this position, with the patient resting on the right side, the degree of plasma leakage may be quantified by means of the pleural effusion index. The pleural effusion index is calculated as 100 times the maximum width of the right pleural effusion, divided by the maximal width of the right hemithorax. Source: Vaughn DW, Green S, Kalayanarooj S, et al. Dengue in the early febrile phase: viremia and antibody responses. J Infect Dis 1997; 176:322-30 (CDC Dengue Slideset).
              • Observed: 14.7% in patients with dengue hemorrhagic fever had pleural effusions or ascites (Malavige et al., 2006).
            • Positive Tourniquet Test:
              • Description: A positive tourniquet test (more than 20 petechiae in a square patch of skin 2.5 x 2.5 cm [greater than 20/in(2)]) may be found in over one-third of patients with DF (Rigau-Perez et al., 1998).


                • Positive Tourniquet Test (CDC Dengue Slideset):



                  Description: This slide demonstrates what a typical positive result from a tourniquet test may look like. This patient has more than 20 petechiae per square inch. Source: CDC (CDC Dengue Slideset)
              • Observed: 76-100% in Thai children with classical dengue hemorrhagic fever (Pan American Health Organization, 1994).
            • Splenomegaly:
            • Thrombocytopenia:
            • Vaginal bleeding:
            • Vomiting:
              • Description: Several symptoms and signs occur before defervescence and may serve as warning signs that DHF and DSS are impending: generalised abdominal pain, persistent vomiting, change in the level of consciousness, a sudden drop in the platelet count, and a rapid rise in the hematocrit (Mairuhu et al., 2004).
              • Observed: 68% in patients with dengue hemorrhagic fever had vomiting (Malavige et al., 2006). 62% of Puerto Ricans cases confirmed with with dengue hemorrhagic fever in the laboratory had vomiting (Malavige et al., 2006).
          • Syndrome -- Dengue Shock Syndrome:
            • Description: The World Health Organisation defines DSS as DHF with circulatory failure as manifested by a rapid, weak pulse with narrowing of the pulse pressure (less than or equal to 20 mmHg, regardless of pressure levels, e.g. 100/90 mmHg) or hypotension with cold, clammy skin and restlessness. In Asia, DHF and DSS mainly affect children under 15 years of age in hyperendemic areas. The age distribution is different in the Americas, where these syndromes occur in all age groups. However, the majority of fatalities during epidemics in the Americas occur in children (Mairuhu et al., 2004). In severe cases, the patient's condition suddenly deteriorates after a few days of fever. At the time of or shortly after the temperature drop, between 3 and 7 days after onset, there are signs of circulatory failure: the skin becomes cool, blotchy, and congested; circumoral cyanosis is frequently observed, and the pulse becomes weak and rapid. Although some patients may appear lethargic, they become restless and then rapidly go into a critical stage of shock. Acute abdominal pain is a frequent complaint shortly before the onset of shock (Pan American Health Organization, 1994). The liver may be palpable and tender; and liver enzymes are usually mildly abnormal but jaundice is rare. The four warning signs for impending shock are intense, sustained abdominal pain; persistent vomiting; restlessness or lethargy; and a sudden change from fever to hypothermia with sweating and prostration. The development of any of these signs or any suggestion of hypotension are indications for hospital admission and management to prevent shock. The patient may recover rapidly after volume replacement but shock may recur during the period of excessive capillary permeability. The prognosis in DHF/DSS depends on prevention or early recognition and treatment of shock. In hospitals with long experience of DSS the case fatality rate in DHF can be as low as 0.2%. Once shock has set in the fatality rate may be high (12% to 44%) (Rigau-Perez et al., 1998).
            • Observed: 26-50% in Thai children with classical dengue hemorrhagic fever developed shock (Pan American Health Organization, 1994). 18.7% in patients with dengue hemorrhagic fever developed shock (Malavige et al., 2006).
          • Syndrome -- Dengue Fever-Unusual Manifestations:
            • Description: An increasing number of dengue infections have been related to other unusual manifestations. These include dengue fever with severe haemorrhage, fulminant liver failure, cardiomyopathy, and neurological phenomena such as altered consciousness, convulsions, and coma resulting from enchephalitis and encephalopathy. Previously, neurological manifestations were ascribed to nonspecific complications secondary to DHF and DSS. Possible causes of dengue encephalopathy include hypotension, cerebral oedema, focal haemorrhage, hyponatraemia, and fulminant hepatic failure. However, a recently documented possibility is the invasion of the central nervous system. Other unusual presentations include ocular manifestations (Mairuhu et al., 2004).

        6. Treatment Information:
          • Suppportive Treatment: Treatment is supportive and includes bed rest, anitpyretics, and analgesics. In case of dehydration, fluid and electrolyte replacement are used in addition (Burke and Monath, 2001). Ribavirin has marginal value (Burke and Monath, 2001).

    4. Prevention:
      1. Environmental Management-Naturalistic Methods:
        • Description: Naturalistic methods involve changes to the natural environment designed to suppress the abundance of immature stages of vector mosquitoes. These measures may be either long-term, which are based on filling or draining of potential aquatic breeding sites for the vector, or short-term. On occasion, A. aegypti may breed in newly-constructed or abandoned septic tanks or latrines, and this may be prevented by draining or filling. More temporary naturalistic measures may include landscaping efforts to remove the vegetation that provides shade, food, or water collection that might contribute to the abundance of these vector mosquitoes; vegetation near the home may influence the abundance of A. aegypti. Where possible, brush should be cut back or removed from the immediate vicinity of homes. Treeholes and other natural rainwater receptacles should be filled with concrete, sand, packed earth, gravel, or other suitable materials. Stumps and other vegetation near houses that could become foci should be removed, or at least cut back annually (Pan American Health Organization, 1994).
      2. Improved Domestic Water Supply:
        • Description: One of the keys to the control of urban Aedes vectors, particularly A. aegypti, is improved domestic water supply (Pan American Health Organization, 1994). Potable water must be delivered in sufficient quantity, quality, and consistency year-round in order to reduce the use of major breeding sites, duchas, drums, overhead tanks, and jars. Individual household piped water supplies are the preferred alternative to the use of wells, communal standpipes, rooftop catchments, and other water delivery systems (Pan American Health Organization, 1994).
        • Efficacy:
          • Rate: We investigated the hypothesis that a deficient supply of piped water was causing a high prevalence of water storage containers, which in turn, become important aquatic habitats of Aedes aegypti in a small town in Venezuela. The House (71.2%) and Breteau indices (229) were considerably elevated. Prevalent positive containers were: metal drums, small disposable containers (bottles, tins, etc.), tires, house plants (flowers in vases and plants in pots with earth) and tanks. Most people reported frequent interruptions in the supply of piped water and considered it to be unreliable. The frequency of interruptions in the supply of water was positively correlated with the House and Container indices, and with the number of positive containers, water-storage devices and positive water-storage devices per house. Even people who considered that they had an adequate supply of water kept numerous water storage containers. Most people (60%) said they would not stop storing water even in the event of the establishment of a reliable supply of piped water (Barrera et al., 1993).
          • Duration:
      3. Solid Waste Management:
        • Description: Vector control efforts employing solid waste management protect public health and conserve natural resources. Proper storage, collection, and disposal of solid wastes protects the public health, while the reduction of the generation of wastes, reuse, and recycling conserve natural resources. Both approaches require community education and participation. Solid waste management for successful vector control consists of three aspects: waste reduction, recycling and re-use; collection; and appropriate disposal (Pan American Health Organization, 1994).
        • Efficacy:
          • Rate: We established a waste recycling system and promoted a breeding site reduction campaign for waste management, including the application of Temephos in containers to kill larvae. For the drinking water management, fish were released in water containers to prevent larval breeding. It should be mentioned that with the integrated pest control and regular inspections of Aedes larvae in Taiwan the density figures 1, 2-5, and 6 or above for Aedes aegypti were 38.7%, 42.9%, and 18.4%, respectively, in 1988, and in 1993 were 90.8%, 9.2% and 0%. The incidence of dengue fever cases has 98% decreased since 1988. In 1990 and 1993, there was no indigenous cases. We have concluded that integrated pest control is the best and most effective method for dengue fever control, including solid waste and drinking water management (Chen et al., 1994).
          • Duration:
      4. Tire Management:
        • Description: Surveys were conducted in some townships along the national highways and trunk roads of northeast India to detect breeding of Aedes mosquitoes in used/waste tire dumps piled outdoors by the tire repairing shops during summer season of 1996-1997. The breeding of both the potential vectors of dengue, viz. Aedes aegypti and Ae. albopictus were detected, prevalence rate being in the range of 30.0-88.0 (CI = container index value). The preponderance of Ae. aegypti was considerably much higher than that of Ae. albopictus and all the urban and semiurban areas coming up along the side of the roads were observed to be infested with Ae. aegypti. With respect to transmission of dengue, this study clearly indicates that waste tire dumps in every urban agglomeration should receive primary attention in view of their relative contribution to the abundance and dispersal of these vector mosquitoes (Dutta et al., 1998) Waste tire stockpiles are a significant public health (vectors) and safety (fire hazard) concern in cities, and imported used tires are believed responsible for the introduction of A. albopictus into the United States. Tires may be treated with insecticides, salt, or soap for chemical control of immature mosquitoes. New technologies for tire reuse and disposal are continually coming into use, but most of them have proved to be of limited application or cost-effectiveness (Pan American Health Organization, 1994).
        • Efficacy:
          • Rate: VectoLex (22.6 kg) was used to treat approximately 6,000 car and truck tires; some of the tires were in direct sunlight whereas others were shaded. Aedes triseriatus was the dominant species in these tires. Tires treated with VectoLex contained significantly fewer mosquitoes than control tires, and even 65 days after application, control tires were 16.7 times more likely to contain larvae (Siegel and Novak, 1999).
          • Duration:
      5. Vector Control-Larvicide-Temephos:
        • Description: Larviciding of "focal" control of A. aegypti is usually limited to domestic-use containers that cannot be destroyed, eliminated, or otherwise managed. There are three insecticides that can be used for treating containers that hold drinking water: One percent temephos (Abate) sand granules applied to the containers using a calibrated plastic spoon to administer a dosage of 1 ppm. This dosage has been found to be effective for 8-12 weeks; The insect growth regulator methoprene (Altosid) is used in the form of briquettes; BTI (Bacillus thuringiensis H-14) (Pan American Health Organization, 1994).
        • Efficacy:
          • Rate: Trial tests and container observations were conducted in households to verify the residual effect of temephos in Manaus, Amazonas State, Brazil. Three plastic buckets, three tin cans, and three tires filled with water from an artesian well and larvicide were used in the experiment, with twenty-five third-instar larvae, which remained exposed for 24h, followed by mortality readings. The same types of containers were selected from common households. Collection and counts followed by chemical treatment were carried out on the larvae that were found. Follow-up was performed weekly to verify recolonization by Aedes aegypti. The experiment showed 100% mortality in the plastic buckets until day 90, and 80% in the tin cans until day 30, decreasing from day 45 onwards. Mortality in the tires decreased to 35% in the first month. Household results showed 100% mortality for all containers after 24h and differentiated values in the subsequent readings. Larvae were observed on day 35 in a tin can and on day 21 in a gallon can. There was a large diversity of results in the tires, with recolonization observed from day 7 onwards (Pinheiro and Tadei, 2002).
          • Duration: This dosage has been found to be effective for 8-12 weeks (Pan American Health Organization, 1994).
      6. Vector Control-Larvicide-Methroprene:
        • Description: Larviciding of "focal" control of A. aegypti is usually limited to domestic-use containers that cannot be destroyed, eliminated, or otherwise managed. There are three insecticides that can be used for treating containers that hold drinking water: One percent temephos (Abate) sand granules applied to the containers using a calibrated plastic spoon to administer a dosage of 1 ppm. This dosage has been found to be effective for 8-12 weeks; The insect growth regulator methoprene (Altosid) is used in the form of briquettes; BTI (Bacillus thuringiensis H-14) (Pan American Health Organization, 1994). Methoprene-derived mortality occurs mainly at the pupa stage and pupa development is inversely proportional to methoprene concentration (Braga et al., 2005).
        • Efficacy:
          • Rate: The tank bromeliad Billbergia pyramidalis was treated with 2 doses (0.5 and 2 g) of ALTOSID Granules or Pellets for the control of Aedes aegypti L. Emergence inhibition (EI) for all mosquito pupae (including natural populations) in the center wells and leaf axils was greater than 90% for at least 6 and 12 months for both doses of granules and pellets, respectively. No significant difference in %EI was found between center wells and leaf axils (Ritchie and Broadsmith, 1997).
          • Duration:
      7. Vector Control-Space Sprays:
        • Description: Space spraying involves the application of small droplets of insecticide into the air in an attempt to kill adult mosquitoes (Pan American Health Organization, 1994). Pant and Yasuno demonstrated that 95% of A. aegypti rest indoors, and of these, greater than 90% do so on surfaces that could not be sprayed with residual compounds. Therefore the intervention commonly used during epidemics was and still is, the ground application of small quantities of an aerosol insecticide in gas oil or kerosene as carrier (ultra low volume (ULV) (Seccacini et al., 2006).
        • Efficacy:
          • Rate: Unfortunately many of the campaigns to reduce vector populations have not been successful due to practical problems associated with the treatments of densely populated areas, lack of funds and failure to sustain implementation of control programs as well as irritation problems in the inhabitants associated with the way compounds were applied (Seccacini et al., 2006).
          • Duration:
      8. Biological Vector Control-Fish:
        • Description: Various fish can be used to eliminate mosquitoes from larger containers used to store potable water. These include Gambusia affinis and Poecilia spp. These and other fish may be potentially used where their introduction can be maintained and where the human population does not object to the presence of such obvious organisms in their domestic water-storage containers. Local, endemic larvivorous fish species also may be evaluated for efficacy against A. aegypti larvae (Pan American Health Organization, 1994).
      9. Biological Vector Control-Toxorhynchites:
        • Description: Although various larval mosquitoes feed on other mosquitoes, Toxorhynchites mosquitoes have two advantages as predators: they develop in the same kinds of containers as A. aegypti and they do not feed on blood. Field trials have produced mixed results. On Union Island in Saint Vincent and the Grednadines, adult emergence was reduced when larval Toxorhynchites were introduced by hand, but the effect on adult abundance was not recorded. In Indonesia, sustained release of first instar predatory larvae into virtually all household water storage basins failed to reduce the abundance of adult A. aegypti. This lack of effect may have been due to an inability of first instar Toxorhynchites larvae to survive in the absence of small prey (Pan American Health Organization, 1994).
        • Efficacy:
          • Rate:
          • Duration: Each week, fifty Ae. aegypti first instar larvae were introduced to each of five water-filled drums (220 litres) of the type commonly used for domestic water storage in Caribbean dwellings. At the beginning of the fourth week, a certain number (0, 1, 2, 5 or 10) of first instar Tx. moctezuma larvae were introduced to each drum and the daily yield of Ae. aegypti adults from each drum was monitored thereafter. The experiment was repeated three times. With only one or two Tx. moctezuma larvae, predation on Ae. aegypti larvae stopped the output of Ae. aegypti adults for 1 week. Five or ten Tx. moctezuma prevented any Ae. aegypti emergence for up to 16 weeks (Tikasingh, 1992).
      10. Biological Vector Control-Hormone Mimics:
        • Description: The juvenile hormone (JH) analog methoprene is a larvicide that can be substituted for temephos in localities where Ae. aegypti exhibits resistance to this organophosphate (Braga et al., 2005). Methoprene now promises to be the most environmentally acceptable larvicidal chemical usable against mosquitoes. The apparent absence of mammalian toxicity of this synthetic juvenile hormone permits its use in portable water (Pan American Health Organization, 1994). Susceptibility of the Brazilian Ae. aegypti populations to methoprene alone suggests this insect growth regulator could substitute for temephos in the control of the dengue vector in the country (Braga et al., 2005).
      11. Biological Vector Control-Cyclopoids:
        • Description: Certain ubiquitous cyclopoid copepods ("water fleas") prey on newly hatched larvae. If the mosquitoes develop to the third instar, however, they are too large to be attacked by these minute predators (Pan American Health Organization, 1994). Recently, copepods have emerged as a new tool for controlling mosquito vectors, particularly those inhabiting artificial containers. In Tahiti, Mesocyclops leuckarti (Claus) and Mesocyclops aspericornis (Daday) were successfully used against the dengue vector Aedes aegypti (L.) and in Honduras this mosquito was suppressed in peridomestic containers by Mesocyclops longisetus (Thiebaud). In New Orleans, Macrocyclops albidus (Jurine) eliminated Aedes albopictus (Skuse) from tire piles in a wooded area. More recently, in Vietnam, Mesocyclops sp. succeeded in eliminating Ae. aegypti (Dieng et al., 2006)
        • Efficacy:
          • Rate: When copepods were present, the immature stages of Ae. albopictus were nearly eliminated. Macrocyclops and the mixture of three genera were the most effective in reducing the older instars of Ae. albopictus. Although predation pressure was high, a few Ae. albopictus pupated in all the treated containers (Dieng et al., 2006).
          • Duration:
        • Complication: Although these copepods suppressed Ae. albopictus in the 500 mL containers, this mosquito uses a wide variety of artificial containers as larval habitats and prefers to colonize cryptic microhabitats. Such behaviour makes it difficult to monitor all the productive sites. However, treatment of most of the artificial containers with predatory copepods such as those studied here could possibly reduce adult Ae. albopictus populations in our immediate environment (Dieng et al., 2006).
      12. Biological Vector Control-Bacillus thuringiensis:
        • Description: Bacillus thuringiensis israelensis (BTI), which was discovered in the 1970s, is a proven, environmentally nonintrusive mosquito larvicide that appears to be entirely safe for people. This fermentation product of Bacillus thruingiensis H-14 is the most acceptable material currently on hand for use against mosquitoes. It has become commercially available under such trade names as Teknar, Vectobac, and Bactimos, and can be purchased in lots of up to one-quarter of a million pounds (Pan American Health Organization, 1994). VectoBac DT, a tablet formulation of Bacillus thuringiensis israelensis (Bti) was evaluated for the potential control of dengue vectors in various types of potable water containers. On introduction to containers, the tablet sinks to the bottom and the Bti toxins are found concentrated at the sides and the base, while the treated water column is free of Bti toxins within 24 hours after tablet introduction. In a simulated study, earthen, HDPE and plastic containers were kept covered and laboratory-bred larvae were introduced to determine the control by the tablet (Benjamin et al., 2005).
        • Efficacy:
          • Rate: The efficacy and persistence of the tablet, with a control of greater than 90%, was significantly longer in earthen containers in comparison to the HDPE and plastic containers. Efficacy and persistence were observed in earthen containers for a minimum period of 5.5 months (166 days) both without water replenishment and with weekly, 50% water volume, replenishment, and for a maximum period of 2.2 months (66 days) with daily, 50% water volume, replenishment. In plastic and HDPE containers, the tablet activity had a persistence of 2.1 months (63 days) without water replenishment and 1.8 months (54 days) with weekly water replenishment. The efficacy and persistence of the VectoBac DT was significantly longer in the earthen containers, with or without regularly treated water exchange, due to the Bti toxins being embedded in the porous earthen container surfaces, which protects them from rapid degradation. Lesser toxin amounts are removed from the water column during water exchange. The efficacy of VectoBac DT was also evaluated for the control of natural infestation of Aedes larvae which were resistant to temephos at the WHO diagnostic dosage of 0.012 mg/l. The tablet significantly reduced the pupal density by 8 fold in earthen containers for 67 days and 5 fold in HDPE containers for 55 days in comparison to untreated containers (p less than 0.05). However, the tablet was effective for a shorter period of 25 days post-tablet-introduction due to fungal infestation in the treated plastic containers (Benjamin et al., 2005).
          • Duration:

    5. Model System:
      1. Suckling Mouse Encephalitic Model:
        1. Model Host: Suckling albino mice (Sriurairatna et al., 1973).
        2. Model Pathogens:
        3. Description: The encephalitic model in suckling mouse is very well known and has been extensively used for many years. It shows direct cytopathology of the viruses to the neurons of suckling mice. This, however, is not a model of dengue fever of DHF in humans (Bhamarapravati, 1997). The infected mice showed definite paralysis on day 4 postinoculation with dengue-2 virus, and they were severely paralyzed on day 5. None of the infected mice survived to day 6 (Sriurairatna et al., 1973).
      2. Primate Model:
        1. Model Host: Monkeys and chimpanzees (Bhamarapravati, 1997). Chimpanzees, gibbons, and macaques (Gubler, 1988).
        2. Description: Monkeys and chimpanzees infected with dengue viruses develop viremia and specific antibodies against dengue viruses, but they show no clinical disease (Bhamarapravati, 1997). Experimental laboratory evidence shows that several species of lower primates (chimpanzees, gibbons, and macaques)become infected and develop viremia titers sufficient to infect mosquitoes. Onset of viremia after infection in these animals is similar to that in humans, but the magnitude and duration of viremia generally are lower and shorter, respectively (Gubler, 1988).
      3. Mouse Model of DHF/DSS-like Manifestations:
        1. Model Host: Female SCID (C.B.-17/Icr Tac-scid) mice (An et al., 1999). To develop a useful animal model for DEN virus infection, HepG2 cells, which support DEN viral replication, were transplanted into SCID mice (An et al., 1999).
        2. Model Pathogens:
        3. Description: An elegant mouse model for dengue virus infection has now been described, which may be useful in studying various aspects of the pathogenesis of DHF. A human hepatocarcinoma cell line, HepG2, known to support dengue virus replication was transplanted into severe combined immunodeficient (SCID) mice. Such HepG2-grafted mice were infected with DEN-2 virus, and the authors showed very convincingly that the mice developed many of the features of DHF, including thrombocytopenia and increased haematocrit. Virus inoculated intraperitoneally was detected in the serum and in the liver, and also induced paralysis, at which point virus was also detected in the brain (Cardosa, 2000).
  2. Nonhuman Primates:
    1. Taxonomy Information:
      1. Species:
        1. Bonnet macaque :
          • GenBank Taxonomy No.: 9548
          • Scientific Name: Macaca radiata (NCBI Taxonomy)
          • Description: To investigate the ecology of dengue and Japanese encephalitis (JE) viruses in the forest in Asia, a seroepidemiological survey was carried out on 358 Southeast Asian cynomologus (Macaca iris), 33 Indian bonnet (Macaca radiata) and 37 Japanese (Macaca fuscata) monkey sera by a plaque reduction neutralization test. The results indicated that Southeast Asian monkeys were naturally infected with these viruses but the frequency of antibody to them varied considerably according to the geographical origin of the monkeys (Yuwono et al., 1984).
        2. Crab-eating macaque :
          • GenBank Taxonomy No.: 9541
          • Scientific Name: Macaca fascicularis (NCBI Taxonomy)
          • Description: Yuwono et al. reported that out of 74 cynomolgus monkeys (Macaca iris) in the Philippines, 14.9% were positive for DEN and 2.7% were positive for JE, as evidenced by a plaque reduction neutralization test (Inoue et al., 2003). To investigate the ecology of dengue and Japanese encephalitis (JE) viruses in the forest in Asia, a seroepidemiological survey was carried out on 358 Southeast Asian cynomologus (Macaca iris), 33 Indian bonnet (Macaca radiata) and 37 Japanese (Macaca fuscata) monkey sera by a plaque reduction neutralization test. The results indicated that Southeast Asian monkeys were naturally infected with these viruses but the frequency of antibody to them varied considerably according to the geographical origin of the monkeys (Yuwono et al., 1984). Synonyms: Macaca irus, Macaca cynomolgus (NCBI Taxonomy).
        3. Japanese macaque :
          • GenBank Taxonomy No.: 9542
          • Scientific Name: Macaca fuscata (NCBI Taxonomy)
          • Description: To investigate the ecology of dengue and Japanese encephalitis (JE) viruses in the forest in Asia, a seroepidemiological survey was carried out on 358 Southeast Asian cynomologus (Macaca iris), 33 Indian bonnet (Macaca radiata) and 37 Japanese (Macaca fuscata) monkey sera by a plaque reduction neutralization test. The results indicated that Southeast Asian monkeys were naturally infected with these viruses but the frequency of antibody to them varied considerably according to the geographical origin of the monkeys (Yuwono et al., 1984).
        4. Mitred leaf monkey :
          • GenBank Taxonomy No.: 78451
          • Scientific Name: Presbytis melalophos (NCBI Taxonomy)
          • Description: Of 114 monkeys tested by hemagglutinatin inhibition (HI), 82.5% had dengue antibody and only 25.9% had Japanese encephalitis (JE) antibody at a serum titer of 1:20 or higher. Of 233 monkeys, including the same 114 tested by HI, 62.8% had dengue neutralizing antibody of 2 logs or greater, while only 2 of 46 tested had JE neutralizing antibody (4.3%) (Rudnick, 1965). It appears reasonable to consider that most of the high-titered dengue neutralizing antibody (3.0 logs or greater) in monkeys is actually a result of dengue infection (Rudnick, 1965). P. melalohos-HI for Dengue, 3 positives out of 4 total tested, 66.7% positive. P. melalohos-NI for Dengue, 2 positives out of 2 total tested, 100% positive (Rudnick, 1965).
        5. Orangutan :
          • GenBank Taxonomy No.: 9600
          • Scientific Name: Pongo pygmaeus (NCBI Taxonomy)
          • Description: Between 1996 and 1998, 84 free-ranging orangutans captured for translocation, underwent a complete health evaluation. Analogous data were gathered from 60 semi-captive orangutans in Malaysia. Baseline hematology and serology; vitamin, mineral and pesticide levels; and results of health evaluations, including physical examination, provide a baseline for future monitoring. Free-ranging and semi-captive orangutans shared exposure to 11 of 47 viruses (Kilbourn et al., 2003). There was evidence of exposure to respiratory syncytial virus, coxsackie virus, dengue virus, and zika virus in both groups (Kilbourn et al., 2003). The presence of neutralizing antibodies among wild orangutans strongly suggests the existence of sylvatic cycles for dengue, Japanese encephalitis, and sindbis viruses in North Borneo (Wolfe et al., 2001).
        6. Pig-tailed macaque :
          • GenBank Taxonomy No.: 9545
          • Scientific Name: Macaca nemestrina (NCBI Taxonomy)
          • Description: Of 114 monkeys tested by hemagglutinatin inhibition (HI), 82.5% had dengue antibody and only 25.9% had Japanese encephalitis (JE) antibody at a serum titer of 1:20 or higher. Of 233 monkeys, including the same 114 tested by HI, 62.8% had dengue neutralizing antibody of 2 logs or greater, while only 2 of 46 tested had JE neutralizing antibody (4.3%) (Rudnick, 1965). It appears reasonable to consider that most of the high-titered dengue neutralizing antibody (3.0 logs or greater) in monkeys is actually a result of dengue infection (Rudnick, 1965). M. nemestrina-HI for Dengue, 1 positives out of 2 total tested,50% positive. P. melalohos-NI for Dengue, 1 positives out of 2 total tested, 50% positive (Rudnick, 1965).
        7. Presbytis obscura :
          • GenBank Taxonomy No.: 78450
          • Scientific Name: Presbytis obscura (NCBI Taxonomy)
          • Description: The studies by Rudnick's group, which had as their objective the demonstration of a sylvatic cycle of dengue, were carried out in forests of various ecologic types, but all were characterized by abundant monkey populations and relative isolation from human populations. Some were characterized by a small human population at the forest edge in the form of a sentinel village and, especially by the complete absence of Aedes aegypti and the presence of Aedes albopictus. These studies led to the isolation of several strains of dengue (types 1, 2 and 4) from sentinel monkeys [Prebytis obscura and Macaca fascicularis (=irus)] placed in the forest canopy (Rodhain, 1991). One or more dengue serotypes, transmitted by Aedes of the niveus group circulate in the forest canopy in primeval cycle among certain species of monkeys (Macaca sp. and Presbytis sp.-which have asymptomatic infections) in a silent cycle (Rodhain, 1991).
        8. Silvered leaf monkey :
          • GenBank Taxonomy No.: 122765
          • Scientific Name: Trachypithecus cristatus (NCBI Taxonomy)
          • Description: Of 238 monkey sera tested by HI, the majority represented Macaca irus, the long-tailed macaque, and Presbytis cristatus, the silvered leaf monkey, two of the most common species in Malaya. For all species, the percentage of positives for the four dengue types ranged from 52 to 62% (Rudnick et al., 1967).
        9. Toque macaque :
          • GenBank Taxonomy No.: 9552
          • Scientific Name: Macaca sinica (NCBI Taxonomy)
          • Description: The population of toque macaques used in the current study inhabit the natural dry evergreen forest within the Nature and Archaeological Reserve at Polonnaruwa. The behavior, ecology, demography, and genetics of these wild monkeys have been intensively studied since 1968 by Dittus and others. In 1995, the population comprised nearly 1,000 monkeys distributed among some 28 social groups. All the macaques in the population have been individually identified and the dates of birth and life histories of nearly all the animals are known. The ages of animals whose births were not observed, such as immigrants into the study population, were estimated based on known relationships between morphologic development and age (de Silva et al., 1999). Two hundred forty-four serum samples collected between July and October 1995 were screened for the presence of antibodies against dengue-2 virus. Twenty-one percent (52 of 244) of the animals tested positive for dengue virus antibody (de Silva et al., 1999).

    2. Infection Process:

      No infection process information is currently available here.

    3. Disease Information:

      No disease information is currently available here.

    4. Prevention:

      No prevention information is currently available here.

    5. Model System:

      No model system information is currently available here.

  3. Other Mammals:
    1. Taxonomy Information:
      1. Species:
        1. Armadillo :
          • GenBank Taxonomy No.: 9360
          • Scientific Name: Dasypus spp (NCBI Taxonomy).
          • Description: Dengue seroneutralizing antibodies were found in five species: Armadillo, porcupine, opossum, agouti and brocket deer. The role of free-ranging species in the maintenance of DENV in the wild in South America is disputed. Nevertheless, it is unlikely that another flavivirus induced the seroneutralization reaction seen, since DENV makes its own antigenic complex within the Flaviviridae. After Platt et al. reported neutralizing antibodies to DENV in bats, we found evidence for infection of a very large number of diverse forest neotropical mammals, indicating that wild animals could be exposed to DENV, once it has been introduced into a pristine area, possibly consecutive to human activities, such as tourism, hunting, logging, and gold mining. The exact role of accidentally exposed species is not known, but they may act as temporary reservoirs, with transmission by either forest populations of A. aegypti (de Thoisy et al., 2004) 60 Dasypus spp tested, 3 positive for Dengue 2 (de Thoisy et al., 2004).
        2. Brown four-eyed opossum :
          • GenBank Taxonomy No.: 42725
          • Scientific Name: Metachirus nudicaudatus (NCBI Taxonomy)
          • Description: Dengue seroneutralizing antibodies were found in five species: Armadillo, porcupine, opossum, agouti and brocket deer (de Thoisy et al., 2004). 19 Metachirus nudicaudatus tested, 1 positive for Dengue 2 (de Thoisy et al., 2004).
        3. Brazilian agouti :
          • GenBank Taxonomy No.: 42152
          • Scientific Name: Dasyprocta leporina (NCBI Taxonomy)
          • Description: Dengue seroneutralizing antibodies were found in five species: Armadillo, porcupine, opossum, agouti and brocket deer (de Thoisy et al., 2004). 29 Dasyprocta leporina tested, 1 positive for Dengue 2 (de Thoisy et al., 2004).
        4. Porcupines :
        5. Brocket deer :

    2. Infection Process:

      No infection process information is currently available here.

    3. Disease Information:

      No disease information is currently available here.

    4. Prevention:

      No prevention information is currently available here.

    5. Model System:

      No model system information is currently available here.

  4. Aedes mosquitoes:
    1. Taxonomy Information:
      1. Species:
        1. Yellow fever mosquito :
          • GenBank Taxonomy No.: 7159
          • Scientific Name: Aedes aegypti (NCBI Taxonomy)
          • Description: Both Yellow Fever (YF) and Dengue (DEN) viruses can be transmitted in an urban cycle between humans by the highly domesticated Aedes aegypti mosquito. This species is a very efficient epidemic vector of both viruses because of its close association with humans in urban settings, and its blood-feeding behavior of taking blood from multiple human hosts during a single gonotrophic cycle. The DEN viruses are unique in that they are the only known arboviruses that have fully adapted to humans and are maintained in large urban centers of the tropics in an Ae. aegypti-human-Ae. aegypti cycle without apparent input from the enzootic cycles (Gubler, 2004) Stegomyia aegypti (NCBI Taxonomy)
        2. Aedes africanus :
          • Scientific Name: Aedes africanus (Rodhain and Rosen, 1997)
          • Description: While the vectors involved in transmission from monkey to monkey, and possibly, from monkey to man are not known with certainty, certain species are suspected in view of the isolation of dengue viruses from them and of their biology (i.e. their contact with both monkeys and man, at least in certain ecosystems). In Africa, these species have already been implicated in the transmission of yellow fever, and include the subgenera Stegomyia (Ae. luteocephalus, Ae. africanus and Ae. opok) and Diceromyia (Ae. taylori and Ae. furcifer) (Rodhain and Rosen, 1997).
        3. Asian tiger mosquito :
          • GenBank Taxonomy No.: 7160
          • Scientific Name: Aedes albopictus (NCBI Taxonomy)
          • Description: The mosquito Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae), originally indigenous to South-east Asia, islands of the Western Pacific and Indian Ocean, has spread during recent decades to Africa, the mid-east, Europe and the Americas (north and south) after extending its range eastwards across Pacific islands during the early 20th century. The majority of introductions are apparently due to transportation of dormant eggs in tyres. Among public health authorities in the newly infested countries and those threatened with the introduction, there has been much concern that Ae. albopictus would lead to serious outbreaks of arbovirus diseases (Ae. albopictus is a competent vector for at least 22 arboviruses), notably dengue (all four serotypes) more commonly transmitted by Aedes (Stegomyia) aegypti (L.). Results of many laboratory studies have shown that many arboviruses are readily transmitted by Ae. albopictus to laboratory animals and birds, and have frequently been isolated from wild-caught mosquitoes of this species, particularly in the Americas (Gratz, 2004). Ae. albopictus can be unequivocally incriminated as a vector of dengue only where transmission occurs in the absence of Ae. aegypti or any other potential vector. Such transmission in the absence of Ae. aegypti or other species of Stegomyia, has been seen to occur in parts of China, at one time in Japan and the Seychelles, most recently in Hawaii and possibly La Reunion Island in the Indian Ocean. In other areas, particularly in South-east Asia, it appears that Ae. albopictus serves primarily as a maintenance vector of dengue in rural areas (Gratz, 2004).
        4. Aedes cooki :
          • Scientific Name: Aedes cooki (Rodhain and Rosen, 1997)
          • Description: Usually, a species has come under suspicion because it was the only plausible vector present during a dengue epidemic when Ae. aegypit was either absent or so localized that it could not have been responsible for all transmission. Thus, Ae. polynesiensis (in French Polynesia, the Cook Islands, and Futuna) and Ae. scutellaris (in new Guinea) have been shown to be vectors both by epidemiologic and transmission studies to man or monkeys. Others are suspect based on epidemiologic studies and laboratory infection (Ae. cooki on Niue) or epidemiologic studies alone (Ae. hebrideus on Espiritu Santo in Vanuatu, or Ae. rotumae on Rotuma Island) (Rodhain and Rosen, 1997).
        5. Aedes furcifer :
          • GenBank Taxonomy No.: 299627
          • Scientific Name: Aedes furcifer (NCBI Taxonomy)
          • Description: In west Africa, many mosquitoes in the Aedes subgenera Stegomyia and Diceromyia, most notably Ae. furcifer, Ae. luteocephalus, and Ae. aegypti, are suspected of DENV-2 transmission. Although susceptibility and ability to transmit are not the only important factors determining vectorial capacity, our results improve understanding of the role of each species in DENV-2 transmission cycles and emergence potential. The vectorial role of Ae. furcifer and Ae. luteocephalus, hitherto suspected based on frequent DENV-2 isolations in nature, is supported by their high susceptibility to infection. In the light of our data and their bionomics, Ae. furcifer and Ae. aegypti are good candidate vector species for domestic DENV-2 transmission. As indicated in previous studies, only Ae. furcifer is a strong candidate for virus exchange between the forest and human habitations. Of the susceptible forest mosquitoes, it is the most common in villages and the only one found infected in a domestic environment. In contrast, Ae. luteocephalus appears to be confined to the forest habitat (Diallo et al., 2005) During 1990, Dengue-2 (DEN-2) virus was isolated fro the first time from mosquitoes (Aedes furcifer, six isolates; Ae. taylori, six isolates; Ae. luteocephalus, seven isolates) during an epidemic in which DEN-2 virus also was isolated from humans (Traore-Lamizana et al., 1994).
        6. Aedes hebrideus :
          • Scientific Name: Aedes hebrideus (Rodhain and Rosen, 1997)
          • Description: Usually, a species has come under suspicion because it was the only plausible vector present during a dengue epidemic when Ae. aegypit was either absent or so localized that it could not have been responsible for all transmission. Thus, Ae. polynesiensis (in French Polynesia, the Cook Islands, and Futuna) and Ae. scutellaris (in new Guinea) have been shown to be vectors both by epidemiologic and transmission studies to man or monkeys. Others are suspect based on epidemiologic studies and laboratory infection (Ae. cooki on Niue) or epidemiologic studies alone (Ae. hebrideus on Espiritu Santo in Vanuatu, or Ae. rotumae on Rotuma Island) (Rodhain and Rosen, 1997).
        7. Aedes hensilli :
          • Scientific Name: Aedes hensilli (Savage et al., 1998)
          • Description: A dengue fever/dengue hemorrhagic fever (DF/DHF) outbreak in Yap State caused by dengue-4 virus was confirmed serologically and by virus isolation from serum samples collected on each of three island groups. Most DF/DHF cases occurred during a three-month period between mid-May and early August 1995. Five fatal cases, three of which were in children between the ages of four and 11, occurred between June 20 and July 26. A serosurvey conducted in late August revealed anti-dengue IgM prevalence rates of 18% on Yap, 36% on Eauripik, and 6% on Woleai. The majority of residents (93-100%) on the three islands were positive for anti-dengue IgG antibodies, indicating widespread exposure to dengue viruses. The IgG titers indicative of secondary antibody response were noted on Eauripik (6.5%) and Woleai (17%), but were rare on Yap (0.7%). Entomologic investigations implicated the native mosquito species, Aedes hensilli, a member of the Scutellaris Group of Aedes (Stegomyia), as a previously unrecognized epidemic vector of dengue viruses. Aedes hensilli was the most abundant and widespread member of Ae. (Stegomyia) in Yap State, the only species of Ae. (Stegomyia) on Woleai, and the only mosquito species present on Eauripik (Savage et al., 1998)
        8. Aedes luteocephalus :
          • GenBank Taxonomy No.: 299629
          • Scientific Name: Aedes luteocephalus (NCBI Taxonomy)
          • Description: In west Africa, many mosquitoes in the Aedes subgenera Stegomyia and Diceromyia, most notably Ae. furcifer, Ae. luteocephalus, and Ae. aegypti, are suspected of DENV-2 transmission. Although susceptibility and ability to transmit are not the only important factors determining vectorial capacity, our results improve understanding of the role of each species in DENV-2 transmission cycles and emergence potential. The vectorial role of Ae. furcifer and Ae. luteocephalus, hitherto suspected based on frequent DENV-2 isolations in nature, is supported by their high susceptibility to infection. In the light of our data and their bionomics, Ae. furcifer and Ae. aegypti are good candidate vector species for domestic DENV-2 transmission. As indicated in previous studies, only Ae. furcifer is a strong candidate for virus exchange between the forest and human habitations. Of the susceptible forest mosquitoes, it is the most common in villages and the only one found infected in a domestic environment. In contrast, Ae. luteocephalus appears to be confined to the forest habitat (Diallo et al., 2005) During 1990, Dengue-2 (DEN-2) virus was isolated fro the first time from mosquitoes (Aedes furcifer, six isolates; Ae. taylori, six isolates; Ae. luteocephalus, seven isolates) during an epidemic in which DEN-2 virus also was isolated from humans (Traore-Lamizana et al., 1994).
        9. Aedes mediovittatus :
          • Scientific Name: Aedes mediovittatus (Rodhain and Rosen, 1997)
          • Description: We have circumstantial evidence that Ae. mediovittatus was responsible for transmission of the dengue 5 epidemic in at least 1 rural community of Puerto Rico (San Juan laboratories, unpubl data). Collectively, the data lead us to believe that Ae. mediovittatus may be playing an important role in the maintenance of dengue viruses in Puerto Rico during interepidemic periods. This could explain how viruses like dengue 2 and possibly dengue 3 persisted in a limited island population for over 10 years (Gubler et al., 1985). Aedes (Gymnometopa) mediovittatus is a forest mosquito that has become adapted to the rural peri-domestic environment in the Caribbean region (Puerto Rico, Cuba and other islands). It can be considered a potential vector of dengue in view of its anthropophilia and high degree of susceptibility to infection in the laboratory (Rodhain and Rosen, 1997).
        10. Aedes niveus :
          • Scientific Name: Aedes niveus (Inoue et al., 2003)
          • Description: The sylvatic transmission cycle of DEN viruses among forest monkeys by Ae. niveus is indicated, based on epidemiological studies in the Peninsula Malaya (Inoue et al., 2003). While the vectors involved in transmission from monkey to monkey, and possibly, from monkey to man are not known with certainty, certain species are suspected in view of the isolation of dengue viruses from them and of their biology (i.e. their contact with both monkeys and man, at least in certain ecosystems). In Africa, these species have already been implicated in the transmission of yellow fever, and include the subgenera Stegomyia (Ae. luteocephalus, Ae. africanus and Ae. opok) and Diceromyia (Ae. taylori and Ae. furcifer). In Asia, the suspected vectors of forest dengue are Ae. albopictus and Aedes of the niveus group of the subgenus Finlaya (Rodhain and Rosen, 1997).
        11. Aedes opok :
          • Scientific Name: Aedes opok (Rodhain and Rosen, 1997)
          • Description: While the vectors involved in transmission from monkey to monkey, and possibly, from monkey to man are not known with certainty, certain species are suspected in view of the isolation of dengue viruses from them and of their biology (i.e. their contact with both monkeys and man, at least in certain ecosystems). In Africa, these species have already been implicated in the transmission of yellow fever, and include the subgenera Stegomyia (Ae. luteocephalus, Ae. africanus and Ae. opok) and Diceromyia (Ae. taylori and Ae. furcifer) (Rodhain and Rosen, 1997).
        12. Aedes polynesiensis :
          • GenBank Taxonomy No.: 188700
          • Scientific Name: Aedes polynesiensis (NCBI Taxonomy)
          • Description: Epidemiologic observations have led to the incrimination of several species of Aedes of the subgenus Stegomyia (Ae. aegypti, Ae. albopictus and Ae. polynesiensis) from which virus has been isolated in nature, whose abundance coincides with the incidence of epidemic or endemic dengue, and that are at least partially domestic and anthropophilic (Rodhain and Rosen, 1997). During an outbreak occurring in Futuna (Horne Islands) from October 1976 to January 1977, II strains quite similar to dengue virus type I were isolated from blood of patients in acute phase. Immunitary [sic] responses were noted on 8/12 paired sera submitted to IH test; 4/17 serum samples showed antibody titer presumptive of a recent infection. Entomological survey gave evidence that virus was transmitted by Aedes polynesiensis and confirmed that Futuna is free of A.E. aegypti; other species found were: Culex annulirostris, C. pipiens fatigans, C. sitiens. A viral strain was isolated from Ae. polynesiensis only (Fauran et al., 1978).
        13. Aedes rotumae :
          • Scientific Name: Aedes rotumae (Reed et al., 1977)
          • Description: An explosive epidemic of dengue occurred in Fiji between January and July 1975. All laboratory evidence indicated that type 1 dengue was the only prevalent dengue virus. This type had probably not been in Fiji for 30 years and over 70% of the population was susceptible. Aedes aegypti appeared to be the major vector in urban areas, but circumstantial evidence indicated that Aedes rotumae was a vector in at least one remote area (Reed et al., 1977)
        14. Aedes scutellaris :
          • Scientific Name: Aedes scutellaris (Rodhain and Rosen, 1997)
          • Description: Usually, a species has come under suspicion because it was the only plausible vector present during a dengue epidemic when Ae. aegypit was either absent or so localized that it could not have been responsible for all transmission. Thus, Ae. polynesiensis (in French Polynesia, the Cook Islands, and Futuna) and Ae. scutellaris (in new Guinea) have been shown to be vectors both by epidemiologic and transmission studies to man or monkeys (Rodhain and Rosen, 1997).
        15. Aedes taylori :
          • GenBank Taxonomy No.: 299628
          • Scientific Name: Aedes taylori (NCBI Taxonomy)
          • Description: During 1990, Dengue-2 (DEN-2) virus was isolated for the first time from mosquitoes (Aedes furcifer, six isolates; Ae. taylori, six isolates; Ae. luteocephalus, seven isolates) collected during an epidemic in which DEN-2 virus also was isolated from humans (Traore-Lamizana et al., 1994). While the vectors involved in transmission from monkey to monkey, and possibly, from monkey to man are not known with certainty, certain species are suspected in view of the isolation of dengue viruses from them and of their biology (i.e. their contact with both monkeys and man, at least in certain ecosystems). In Africa, these species have already been implicated in the transmission of yellow fever, and include the subgenera Stegomyia (Ae. luteocephalus, Ae. africanus and Ae. opok) and Diceromyia (Ae. taylori and Ae. furcifer) (Rodhain and Rosen, 1997).
        16. Ochlerotatus triseriatus :
          • GenBank Taxonomy No.: 7162
          • Scientific Name: Ochlerotatus triseriatus (Rodhain and Rosen, 1997)
          • Description: Another possible vector is Ae. triseriatus, of the subgenus Protomacleaya, which has been shown to be capable of transmitting dengue virus in the laboratory. There is, as yet, no evidence that it does so in nature (Rodhain and Rosen, 1997). Synonym: Aedes triseriatus (NCBI Taxonomy).

    2. Infection Process:

      No infection process information is currently available here.

    3. Disease Information:

      No disease information is currently available here.

    4. Prevention:

      No prevention information is currently available here.

    5. Model System:

      No model system information is currently available here.


IV. Labwork Information

A. Biosafety Information:
  1. General biosafety information :
    • Biosafety Level: 2 (CDC Office of Health and Safety (OHS))
    • Applicable: Biosafety Level 2 is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs from BSL-1 in that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists; (2) access to the laboratory is limited when work is being conducted; (3) extreme precautions are taken with contaminated sharp items; and (4) certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment (CDC: Laboratory Biosafety Level Criteria).
B. Culturing Information:
  1. Mosquito Cell Culture for Dengue Virus Isolation :
    1. Description: Mosquito cell culture is the most recent methodology developed for dengue virus isolation. Three cell lines of comparable sensitivity are frequently used, but the most widely used is the C6/36 clone of A. albopictus cells. The use of this cell lineage has provided a rapid, sensitive and economical method for dengue virus isolation. Dengue antigens can be detected in infected cell culture by IFA. This technique is less sensitive than the intrathoracic inoculation of adult mosquitoes, but due to its ability to process several samples at the same time, it has become the standard technique for dengue virus isolation. Compared to the other techniques, the advantages of the mosquito cells are: 1) higher sensitivity than the vertebrate cell lines for the recovery of dengue viruses, 2) they are relatively easy to maintain and grow at room temperature, and 3) it is possible to maintain cultures for up to 14 days without changing the medium. Although some reports describe a cytopathic effect (syncytium formation, presence of multinucleated giant cells) induced by all four serotypes of dengue virus, the cytophatic effect produced in mosquito cell culture by many dengue viruses is difficult to detect, and it can be variable. The cytophatic effect is usually seen when these cells are cultured in tubes (De Paula and Fonseca, 2004). Singh and Paul first succeeded in the maintainence of the four dengue virus serotypes in a mosquito cell line established from larvae of Aedes albopictus. Since then, several other mosquito cell lines have also been used or recommended for dengue virus isolation, such as the AP61 (Aedes pseudoscutellaris), Tra-284 (Toxorynchites amboinensis), C636 (A. albopictus), AP64 (clone of an A. pseudoscutellaris cell line), and CLA-1 (clone of an A. pseudoscutellaris cell line) cell lines (Guzman and Kouri, 1996).

    2. Picture(s):
      1. Mosquito Cell Culture (CDC Dengue Slideset):



        Description: This slide demonstrates the use of mosquito cell cultures to detect dengue virus. The results for this patient's blood sample are positive: the fluorescing cells seen here are infected with dengue virus. Source:CDC (CDC Dengue Slideset).
C. Diagnostic Tests :
  1. Organism Detection Tests:
    1. Intracerebral Inoculation of Newborn Mice:
      1. Time to Perform: unknown
      2. Description: Although all the four serotypes were initially isolated by intracerebral inoculation of suckling mice, this technique has several disadvantages, including high cost, long time for isolation, and low sensitivity. These problems have prevented further recommendation of this methodology for viral isolation (De Paula and Fonseca, 2004). Dengue viruses may infect mice by a number of routes, but the intracerebral route is the most sensitive, especially in 1- to 2-day-old suckling mice, producing paralysis or other signs indicative of pathological involvement of the central nervous system. Unlike most other arthropod-borne viruses, the dengue viruses are not very pathogenic when inoculated into the brain of a newborn mouse, probably since they are not neurotropic. The intracerebral inoculation of newborn mice is currently considered the least sensitive isolation system (Guzman and Kouri, 1996).
    2. Mosquito inoculation with IFA:
      1. Time to Perform: 2-to-7-days
      2. Description: Mosquito inoculation is the most sensitive but the least used method for dengue virus isolation. Four mosquito species have been used: Aedes aegypti, A. albopictus, Toxorhynchities amboinensis and T. splendens. Mosquitoes of both sexes are susceptible. Dengue virus generally replicates to high titers [10(6) to 10(7) MID(50)] in as little as four to five days, depending on the incubation temperature. The detection is made through an indirect immunofluorescence assay (IFA) of mosquito tissues, usually done on the brain or salivary glands. The disadvantages are the hard work required, the need for insectaries to produce large numbers of mosquitoes for inoculation, and the isolation precautions taken to avoid the release of infected mosquitoes (De Paula and Fonseca, 2004). Because of its higher sensitivity, the mosquito inoculation technique is still the method of choice for attempting dengue virus isolation from important specimens and, especially, in fatal cases. A. albopictus and Toxorhynchites splendens have been shown to be useful for dengue virus recovery. A. albopictus mosquitoes have been found to be more sensitive for the detection of dengue viruses than important aim for any laboratory LLCMK2. The use of T. splendens larvae is a more rapid and sensitive method for isolation. However, the high isolation rate obtained with mosquito cell cultures, plus the ability to economically process large numbers of samples, more than makes up for the lower sensitivity of the cell culture system. Additionally, mosquito inoculation requires special facilities to establish the mosquito colonies and a certain degree of technical training (Guzman and Kouri, 1996). Mosquito inoculation is the most sensitive system for dengue virus isolation and both adult and larval mosquitoes can be used. Generally, Toxorhynchites mosquitoes are preferable because of their large size and because they are not haematophagous. Adult male Aedes aegypti and Aedes albopictus mosquitoes are also useful for virus isolation (Guzman and Kouri, 2004).
      3. Picture(s):
        • Mosquito Inoculation (CDC Dengue Slideset):



          Description: This slide shows inoculation of the mosquito Toxorhynchites amboinensis with a patient's serum sample. After 10 days of incubation at 30 C, the mosquito is sacrificed and the virus, if present, is detected by fluorescent antibody testing of the head. Source:CDC (CDC Dengue Slideset).
        • Positive IFA Test (CDC Dengue Slideset):



          Description: Here is a positive result from a fluorescent antibody test. Source:CDC (CDC Dengue Slideset).

  2. Immunoassay Tests:
    1. Hemagglutination-inhibition test (HI):
      1. Time to Perform: unknown
      2. Description: For many years, the HI was the standard method used in dengue virus diagnosis due to its high degree of sensitivity and relatively easy execution. Dengue-specific antibodies are detected for many years (48 years or more), being of great value for seroepidemiological studies and to differentiate primary from secondary infections. In primary infections, acute phase antibodies are detected from the fifth or sixth day of symptoms, usually when antibody titers are above 1:10. The antibody titers of convalescent phase samples are usually below 1:640 in primary infections. On the other hand, in secondary or tertiary infections dengue-specific antibodies are readily detected, and there is a rapid increase of the titer during the first days of the infection, usually to a titer higher than 5:120 . Thus, a titer of 1:1,280 or higher in samples collected during the acute phase or at the beginning of the convalescent phase of the disease is an indication of a dengue secondary infection. The high levels of antibodies remain constant for two to three months in some patients when the titer of antibodies begins to fall. The main disadvantages of the HI test are its lack of specificity, the need for paired samples, and the inability to identify the infecting virus serotype (De Paula and Fonseca, 2004). HI is the accepted serological technique however, as it is time consuming, ELISA has become the most frequently used technique for serological studies (Guzman and Kouri, 2004).
    2. Complement fixation test (CF):
      1. Time to Perform: unknown
      2. Description: The CF is usually not used for routine dengue diagnosis, since it is fairly difficulty to perform, requiring highly qualified and trained personnel to achieve good results. The test is based on the principle that the complement will be consumed during the antigen-antibody reaction. The antibodies detected for CF generally appear later than HI antibodies and they persist for short periods, being of limited value for seroepidemiological studies. They are very specific in the primary infections, contributing to the determination of the infecting serotype, as demonstrated by the monotypic responses observed in primary infections (De Paula and Fonseca, 2004).
    3. Neutralization test (NT):
      1. Time to Perform: unknown
      2. Description: The NT is the most sensitive and specific serological test for dengue virus diagnosis, and it is detected for a long period of time. Due to its high specificity, NT can be used to identify the infecting serotype in primary dengue infections, since a relatively monotypic response is observed in the patients' serum during the convalescent phase. In secondary and tertiary infections, the determination of the infecting serotype by NT is not always reliable. The greatest disadvantages of this method are its high cost, the long time necessary to perform it, and the associated technical difficulties (De Paula and Fonseca, 2004).
    4. ELISA:
      1. Time to Perform: unknown
      2. Description: Up till now, ELISA has been considered the most useful test for dengue diagnosis, due to its high sensitivity and the ease of use. ELISA has been used to detect acute phase (IgM) and convalescent phase (IgG) antibodies, as well as for the detection of antigens (Ag). Since it is easy to perform and there is no need for sophisticated equipment, ELISA has become the most widely used serological method for dengue diagnosis. Also, due to its sensitivity for the detection of acute phase antibodies there is no need for convalescence samples since anti-dengue IgM antibodies appear within five days of the the first clinical symptoms. The IgM production varies considerably among the patients. Some patients will have IgM detectable by the 2nd to the 4th day after the beginning of the symptoms, while others do not develop detectable IgM until the 8th day after disease onset. The IgM antibody titers in primary infections are significantly higher than in secondary infections, although the detection of titers of 1:320 in some cases is not uncommon. The IgM production is much lower and transitory in secondary and tertiary infections. A small percentage of patients have secondary infection with no IgM antibodies detected (De Paula and Fonseca, 2004). Different formats such as capture ELISA, capture ultramicroELISA, dot-ELISA, AuBioDOT IgM capture and dipstick have been developed. Serum, blood on filter paper, and more recently saliva are useful for IgM detection if samples are taken within the appropriate time frame (after five days of onset of fever). Different commercial kits for anti-dengue IgM and IgG detection are available, with variable figures of sensitivity and specificity (Guzman and Kouri, 2004)
    5. MAC-ELISA:
      1. Time to Perform: unknown
      2. Description: Anti-dengue IgM detection using enzyme-linked immunosorbent assay (ELISA) represents one of the most important advances and has become an invaluable tool for routine dengue diagnosis. Specifically, MAC-ELISA (IgM antibody capture ELISA) diagnosis is based on detecting dengue-specific IgM antibodies in the test serum by capturing them using anti-human IgM antibody previously bound on a solid phase. In general, 10% false negative and 1.7% false positive reactions have been observed (Guzman and Kouri, 2004). MAC-ELISA has been found to be much less sensitive than the HI test in paired serum samples collected during acute phase of the disease. The specificity of MAC-ELISA is similar to that of HI in primary infections, as well as in secondary infections, when a monotypic response can be observed, but in general the response is cross-reactive to all dengue serotypes and to other flaviviruses, such as those of Japanese encephalitis, St. Louis encephalitis and yellow fever viruses. In dengue infections, the IgM monotypic response is not correlated with the serotype isolated from patients, and for this reason, MAC-ELISA cannot be used for viral identification. There are several commercial kits available for dengue diagnosis by IgM-capture, including those made by well-known research institutes, such as the Oswaldo Cruz and Pedro Khouri Institutes. MAC-ELISA has been a valuable tool for the surveillance of dengue and DHF/DSS. During epidemics, MAC-ELISA has the advantage of fast detection of the propagation of transmission. In areas where dengue is endemic, MAC-ELISA can be used as a valuable tool in the evaluation of a great number of clinical samples, with relative ease (De Paula and Fonseca, 2004).
    6. IgG-ELISA:
      1. Time to Perform: unknown
      2. Description: IgG-ELISA has been developed and is comparable to the HI test in the sense that it can be used for the differentiation of primary and secondary infections by dengue. The test is simple and easy to do, and it can be used in the analysis of a great number of samples. IgG-ELISA is not very specific, cross-reacting with other flaviviruses, and it is not useful for dengue serotype identification. However, this technique is as highly sensitive as the HI test, and it might be useful in seroepidemiological studies (De Paula and Fonseca, 2004). ELISA for anti-dengue IgG detection is currently widely used for classifying cases based on the kind of infection, primary or secondary. Some protocols use serum dilutions to titer anti-dengue IgG. In others, a ratio of IgM/IgG higher than 1.78 is considered a marker of primary infection, and less is considered a marker of secondary infection (Guzman and Kouri, 2004).

  3. Nucleic Acid Detection Tests: :
    1. RT-PCR Review:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: According to the World Health Organization (WHO), PCR is a powerful method to be used for dengue diagnosis, but it still needs to be better standardized (De Paula and Fonseca, 2004). Aiming at the improvement of the molecular diagnosis of dengue, we have compared three well-established methods of RNA extraction from serum of patients with clinical symptoms of dengue. These methods were based on the QIAamp Viral RNA kit, the Chomczynski-Sacchi technique and TRIzol. The data acquired in this study indicated that the best method of RNA extraction from clinical samples to be used for dengue diagnosis by RT-PCR is the QIAamp Viral RNA kit (De Paula and Fonseca, 2004). In another study, we evaluated the detection of dengue virus by RT-PCR in blood, serum, and buffy-coats of 75 IgM-positive serum samples. Out of the 75 samples, 17 were positive for dengue using RT-PCR, and among these samples, 3 were positive in the blood, 14 positive in the serum and 8 positive in the buffy-coat. These results indicated that serum is the best clinical sample for RT-PCR amplification of dengue genomes (De Paula and Fonseca, 2004). The results of several studies suggest that RT-PCR depends on the region of the genome that is chosen to be amplified and on the primers used to achieve this goal. RT-PCR can be type-specific in terms of dengue virus detection, and the detection threshold is usually less than 100 PFUs for all the serotypes (De Paula and Fonseca, 2004). In Brazil, Figueiredo et al. developed a RT-PCR for identification of Brazilian flaviviruses. The genomes of 13 Brazilian flaviviruses, except Bussuquara virus, were amplified by RT-PCR, using universal primers. Analysis of the RT-PCR products gave reproducible results and three distinct amplicon patterns were observed, allowing for correct identification of dengue viruses, as well as the other flaviviruses (De Paula and Fonseca, 2004). Since the WHO still considers PCR an experimental technique for dengue diagnosis, we conducted a validation study of PCR-based diagnosis with clinical samples collected in a region of Brazil where dengue-1 virus has been circulating at a low incidence rate. Viral detection by RT-PCR was evaluated using the sera of patients with clinical diagnosis of dengue, and the results were compared to those obtained by IgM-capture enzyme-linked immunosorbent assay and virus isolation with the same samples. Our results demonstrated that RT-PCR is far more sensitive than virus isolation for clinical samples, allowing for rapid detection of dengue infections (De Paula and Fonseca, 2004). Another important characteristic of PCR is its ability to identify the dengue serotype responsible for the ongoing disease. RT-PCR and restriction enzyme digestion of amplified DNAs have been used in combination, aiming at the development of a fast and simple virus identification method. We developed a nested-PCR, which was followed by restriction enzyme digestion of the amplicons, to differentiate dengue-1 from dengue-2, since at the time of that study these two serotypes were the most prevalent ones in Brazil. The samples were submitted to a nested-PCR amplification, and the amplicons were digested with Kpn I. These results were compared to virus isolation in C6/36 cells and to those obtained by conventional PCR. The use of nested-PCR yielded a three to four-fold increase in the detection rate of dengue virus. All of the amplicons digested by Kpn I identified dengue-1 virus as the infecting strain. These results indicated that nested-PCR provides a high yield of dengue genome amplification, even in the presence of IgM antibodies, and that restriction enzyme digestion rapidly defines the circulating serotype (De Paula and Fonseca, 2004).
    2. Fourplex Real-Time Reverse Transcriptase PCR Assay for Serotype-Specific Detection of Dengue:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: A real-time quantitative nucleic acid amplification assay has been developed to detect viral RNA of a single DEN virus serotype. Each primer-probe set is DEN serotype specific, yet detects all genotypes in a panel of 7 to 10 representative isolates of a serotype. In single reactions and in fourplex reactions (containing four primer-probe sets in a single reaction mixture), standard dilutions of virus equivalent to 0.002 PFU of DEN-2, DEN-3, and DEN-4 viruses were detected; the limit of detection of DEN-1 virus was 0.5 equivalent PFU. Singleplex and fourplex reactions were evaluated in a panel of 40 viremic serum specimens with 10 specimens per serotype, containing 0.002 to 6,000 equivalent PFU/reaction [0.4 to 1.2 x 10(6) PFU/ml]. Viral RNA was detected in all viremic serum specimens in singleplex and fourplex reactions. Thus, this serotype-specific, fourplex real-time reverse transcriptase PCR nucleic acid detection assay can be used as a method for differential diagnosis of a specific DEN serotype in viremic dengue patients and as a tool for rapid identification and serotyping of DEN virus isolates (Johnson et al., 2005). The assay was 100 times more sensitive for DEN-2, DEN-3, and DEN-4 viruses, with limits of detection from 0.0016 to 0.008 equivalent PFU, than for DEN-1 virus, in which the limit of detection was 0.5 equivalent PFU (Johnson et al., 2005).
      3. Primers:
    3. Real time PCR:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The PCR-based methods were more effective in the first few days of infection, whereas the MAC-ELISA became more sensitive 5 or 6 days after disease onset. These results suggest that the best method for dengue diagnosis is a combination of PCR-based and immunological tests. Real-time RT-PCR was more sensitive than the nested RT-PCR approach. Furthermore, it was rapid, reproducible and highly specific, making it a potential method for the diagnosis of dengue fever (de Oliveira Poersch et al., 2005). Real-time RT-PCR was more sensitive, detecting more than four times as many acute-phase samples (collected before the 7th day after disease onset) as the nested RT-PCR. In addition, post-PCR processing and the second round of amplification make the nested RT-PCR protocol time-consuming, prone to false-positive results due to carryover contamination, and subject to misinterpretations caused by the great subjectivity involved in gel electrophoresis analysis. One major problem with RT-PCR assays is the risk of PCR failure due to mismatches in primer and probe binding sites. To avoid false-negative results, multiple genotypes and strains of geographic isolates were considered, and highly conserved regions of the dengue virus genome were chosen for the design of primers and probes. Our serotype-specific assays should, therefore, detect a broad range of dengue virus type 1, 2 and 3 strains. Primers and probes may, nevertheless, be further optimized as novel sequences become available (de Oliveira Poersch et al., 2005).
      3. Primers:

    4. Nucleic Acid Hybridization:
      1. Time to Perform: unknown
      2. Description: Nucleic acid hybridization, using RNA extracted from either dengue virus-infected cell culture supernatants or pools of infected A. albopictus, hybridized either with biotinylated probes or 32P-labelled probes, is used primarily in epidemiological studies. It can also be used for viral diagnosis in tissues obtained in autopsies. The detection method using biotinylated probes is less sensitive than that using radiolabelled probes, and it has not been used for direct viral identification in clinical samples. RNA-RNA hybridization is a sensitive technique that can be applied either directly on fresh samples or on retrospective analyses of fixed samples. Due to the difficulties in working with RNA, as experienced technicians are required to obtain reproducible results, this method has been more often used as a research tool than a routine diagnostic method (De Paula and Fonseca, 2004)

  4. Other Types of Diagnostic Tests:

    No other tests available here.


V. References

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NCBI Taxonomy: Dengue virus type 1 (strain TH-SMAN) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=31633 ].
NCBI Taxonomy: Dengue virus type 1 (strain Western Pacific) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11059 ].
NCBI Taxonomy: Dengue virus type 2 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11060 ].
NCBI Taxonomy: Dengue virus type 2 (isolate Malaysia M1) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11061 ].
NCBI Taxonomy: Dengue virus type 2 (isolate Malaysia M2) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11062 ].
NCBI Taxonomy: Dengue virus type 2 (isolate Malaysia M3) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11063 ].
NCBI Taxonomy: Dengue virus type 2 (NGC-prototype) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11065 ].
NCBI Taxonomy: Dengue virus type 2 (strain 16681) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=31364 ].
NCBI Taxonomy: Dengue virus type 2 (strain 16681-PDK53) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=31635 ].
NCBI Taxonomy: Dengue virus type 2 (strain D2-04) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=31636 ].
NCBI Taxonomy: Dengue virus type 2 (strain Jamaica) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11064 ].
NCBI Taxonomy: Dengue virus type 2 (strain PR159/S1) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11066 ].
NCBI Taxonomy: Dengue virus type 2 (strain PUO-218) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11068 ].
NCBI Taxonomy: Dengue virus type 2 (strain TH-36) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=31637 ].
NCBI Taxonomy: Dengue virus type 2 (strain Tonga 1974) [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11067 ].
NCBI Taxonomy: Dengue virus type 3 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11069 ].
NCBI Taxonomy: Dengue virus type 4 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=11070 ].
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NCBI Entrez Genome: Dengue virus type 2, complete genome [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genome&cmd=Retrieve&dopt=Overview&list_uids=10213 ].
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NCBI Taxonomy: Aedes albopictus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=7160 ].
NCBI Taxonomy: Aedes polynesiensis [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=188700 ].
NCBI Taxonomy: Aedes luteocephalus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=299629 ].
NCBI Taxonomy: Aedes taylori [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=299628 ].
NCBI Taxonomy: Aedes furcifer [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=299627 ].
NCBI Taxonomy: Macaca fascicularis [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=9541 ].
NCBI Taxonomy: Pongo pygmaeus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=9600 ].
NCBI Taxonomy: Ochlerotatus triseriatus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=7162 ].
NCBI Taxonomy: Macaca sinica [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=9552 ].
NCBI Taxonomy: Presbytis obscura [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=78450 ].
NCBI Taxonomy: Trachypithecus cristatus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=122765 ].
NCBI Taxonomy: Macaca fuscata [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=9542 ].
NCBI Taxonomy: Macaca radiata [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=9548 ].
NCBI Taxonomy: Dasypus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=9360 ].
NCBI Taxonomy: Metachirus nudicaudatus [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=42725 ].
NCBI Taxonomy: Dasyprocta leporina [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=42152 ].
NCBI Taxonomy: Coendou [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=43319 ].
NCBI Taxonomy: Mazama [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=43333 ].
NCBI Taxonomy: Presbytis melalophos [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=78451 ].
NCBI Taxonomy: Macaca nemestrina [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=9545 ].
Pacific Center for Emerging Infectious Diseases Research: Dengue 2 virus particles [ hthttp://www.hawaii.edu/pceidr/researchprojects.htm ].
Website 57: Dengue Rapid Strip Test [ http://www.alleight.com/Products/PanBio/dengue_rapid_test.htm ].
D. Thesis References:

No thesis or dissertation references used.


VI. Curation Information